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Development and Characterization of a Controlled Expression
System for Osteogenic Genes
by
Hyun Woo Albert Kim, H. B. Sc.
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Graduate Department of Dentistry
University of Toronto
© Copyright by Hyun Woo Albert Kim, 2011
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Development and Characterization of a Controlled Expression System
for Osteogenic Genes
Hyun Woo Albert Kim
Master of Science Candidate
Graduate Department of Dentistry
University of Toronto
2011
Abstract
Current treatment methods for non-union bone defects present problems. The objective of this
study was to genetically engineer primary and immortalized cell types to express osteogenic
molecules BMP2, RUNX2, OSX, or VEGF in a doxycycline dose-dependent manner for tissue
regeneration. Coding cDNA sequences for all four factors were sub-cloned into the pRTS-1
expression plasmid and transfected into HUCPVCs, RBMCs, ROS cells. Electroporation was the
most effective method of transfection for all cells but stably transfected cells could only be
established for RBMCs and ROS cells. Cells achieved maximum expression within 72hours of
induction and returned to basal levels after 18 days. Enhanced osteogenic bioactivity was only
observed upon activation of BMP-2. The tight regulation of the pRTS-1 system allowed for a
controlled gene expression. Future transplantation experiments using these engineered RBMC
and ROS cells in vivo will evaluate the usefulness of the dox-inducible gene expression system
in bone defects.
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Acknowledgments
For the successful completion of this thesis work, I wish to sincerely thank my professors Dr.
Bernhard Ganss and Dr. John E. Davies. Thank you for your continuous guidance,
encouragement, and most of all, a life-changing opportunity reflected not only by this work, but
also by the way I think as a person. I owe a debt of gratitude for your tireless efforts in correcting
and revising this thesis.
I also wish to thank Mr. James Holcroft for all of his teaching and assistance through the
duration of my graduate program. Your witty personality made our lab into the lively place that I
will never forget. Thank you for always putting a positive spin to my negative results, and also
for keeping my lab bench messy so that I would have something to clean.
Lastly, I would like to acknowledge Dr. Dena Taylor for her efforts in revising and guiding me
through my thesis. Dr. Sharon Zikman for your years of continuous care and support. Thank you
to the members of my committee: Dr. Lidan You, Dr. Seal Peel and Dr. Jane Mitchell for your
suggestions. Many thanks to all the members of the Matrix Dynamics Group for their continuous
support, and to my close friends and family, who have motivated me towards my goal.
This has been a long, but life-defining journey. This opportunity turned some of the most
challenging times of my life into something memorable and rewarding. Looking back, I am very
fortunate to have so many amazing people and positive influences in my life. From the bottom of
my heart, thank you; these lifelong lessons will never be forgotten.
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Table of Contents Abstract ii
Acknowledgments iii
Table of Contents iv
List of Abbreviations vii
List of Tables ix
List of Figures x
Chapter
1.0 Introduction 1
1.1 Non-Union Bone Defects and their Treatment 4
1.2 Tissue Engineering Triad 6
1.2.1 rhBMP-2 Treatment and their Limitations 8
1.3 Methods to Control BMP2 Release 10
1.3.1 rhBMP-2-CBD 10
1.3.2 rhBMP-2-Cell System 10
1.3.3 The Control Mechanism: The Tet-Off and Tet-On Systems 11
1.3.4 pRTS-1: The Enhanced Tet-On System 13
1.3.5 The Cells 16
1.4 Transfection Methods 18
1.5 Rationale 20
1.6 Hypothesis 21
1.7 Objectives 21
2.0 Materials 22
2.1 General Materials 22
2.2 Cloning Materials for pRTS-1-X Constructs 22
2.3 Cell Culture 25
2.4 Kill Curve 28
2.5 Transfections 28
2.6 Transfection Efficiency Analyses 28
2.7 Transcriptional Analyses 28
2.8 Translational Analyses (Western Blots) 29
2.9 Alkaline Phosphatase Activity Assay 31
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3.0 Methods 33
3.1 Cloning of pRTS-1-RUNX2/SP7/VEGF-A Constructs 33
3.1.1 Preparation of pRTS-1 Plasmid Backbone 34
3.1.2 Amplification of the Gene Inserts 37
3.1.3 Ligation of the pRTS-1-X Plasmids 39
3.1.4 Transformation of the pRTS-1-X Plasmids 39
3.1.5 pRTS-1-X Orientation Diagnostic Digest 40
3.2 Cell Culture – General Procedures 41
3.3 HUCPVC Characterization 42
3.4 Hygromycin B Kill Curve of HUCPVC, RBMC, ROS cells 43
3.5 Transfection 44
3.5.1 Amaxa Nucleofector II 44
3.5.2 Invitrogen Lipofectamine 2000 46
3.5.3 Qiagen SuperFect 46
3.5.4 Clonetech Xfect 47
3.5.5 Bio-Rad Gene Pulser MX Cell Electroporation System 47
3.6 Transfection Efficiency Analyses 49
3.7 Single Clone Isolation 49
3.8 mRNA Expression 50
3.9 Western Blot Analyses 51
3.10 Doxycycline Dose-Reponse 52
3.11 Induction Kinetics 52
3.12 Bioactivity Assay 53
4.0 Results 54
4.1 pRTS-1-X Construct Verification 54
4.2 HUCPVC Characterization 55
4.3 Hygromycin B Kill Curve of HUCPVC, RBMC, and ROS cells 56
4.4 Transfection 58
4.4.1 Amaxa Nucleofector II 58
4.4.2 Invitrogen Lipofectamine 2000 66
4.4.3 Qiagen SuperFect 66
4.4.4 Clonetech Xfect 67
4.4.5 Bio-Rad Gene Pulser MX Cell Electroporation System 70
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4.5 Transient vs. Stable Expression and Production of Transfected Clones 72
4.6 Rate of Proliferation 73
4.7 mRNA Expression 74
4.8 Western Blot Analyses 74
4.9 Doxycycline Dose-Response 75
4.10 Induction Kinetics 77
4.11 Alkaline Phosphatase Activity Assay 79
5.0 Discussion 82
5.1 Development of Stable pRTS-1-BMP-2 Cell System is Only Achieved with
Electroporation Combined with RBMCs and ROS Cells
83
5.1.1 Chemical Transfection Methods 85
5.1.2 Electrical Transfection Methods 87
5.1.2.1 Nucleofection 87
5.1.2.2 Electroporation 89
5.2 pRTS-1-BMP-2, Low Background and Doxycycline Dose-Dependent
Activation
90
5.3 pRTS-1 Controlled Kinetics 90
5.4 pRTS-1 Bioactivity 91
5.5 Experimental Limitations 92
Conclusions 95
Future Directions 96
References 97
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List of Abbreviations
Abbreviation Full Description
AB Antibiotics
BM Bone Marrow cells
BMP Bone Morphogenetic Protein
BMP-2 Bone Morphogenetic Protein 2
CBD Collagen type I Binding Domain
Cbfa-1 (also known as RUNX2) Core-binding factor alpha - 1
CMV CytoMegalovirus (promoter)
Dox Doxycycline
EBV Epstein-Barr Virus
ECM Extracellular Matrix
FACS Fluorescence Activated Cell Sorting
eGFP Enhanced Green Fluorescence Protein
GBR Guided Bone Regeneration
GFP Green Fluorescence Protein
GTR Guided Tissue Regeneration
HA Hydroxyapatite
hBM Human Bone Marrow Cells
HIF Hypoxia-Inducible Factor
HUCPVC Human Umbilical Cord Perivascular Cells
MSC Mesenchymal Stem Cell
NEB New England Biolabs
Osx (also known as SP7) Osterix
PCMV CytoMegalovirus promoter
PTet Tetracycline induced Promoter
PTet min. CMV Tetracycline induced cytomegalovirus minimal promoter
pRTS-1-BMP2 pRTS-1 plasmid with the rhBMP-2 gene
pRTS-1-Luc pRTS-1 plasmid with the Luciferase gene
pRTS-1-SP7 pRTS-1 plasmid with the Osterix (sp7) gene
pRTS-1-RUNX2 pRTS-1 plasmid with the RUNX2 gene
pRTS-1-VEGFA pRTS-1 plasmid with the VEGFA gene
pRTS-1-X pRTS-1 plasmid with any of the genes used in this thesis research
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PMT Photomultiplier Tube
RBMC Rat Bone Marrow Cells
rcf Relative Centrifugal Force
ROS Rat Osteosarcoma Cells
rhBMP-2-CBD Recombinant Human Bone Morphogenetic Protein-2-Collagen Type I
Binding Domain
rpm Revolutions Per Minute
RBMC-BMP2 RBMC transfected with pRTS-1-BMP2 construct
RBMC-RUNX2 RBMC transfected with pRTS-1-RUNX2 construct
RBMC-SP7 RBMC transfected with pRTS-1-SP7 construct
RBMC-VEGF-A RBMC transfected with pRTS-1-VEGF-A construct
RE Restriction Enzyme
ROS-BMP2 ROS transfected with pRTS-1-BMP2 construct
ROS -RUNX2 ROS transfected with pRTS-1-RUNX2 construct
ROS -SP7 ROS transfected with pRTS-1-SP7 construct
ROS -VEGF-A ROS transfected with pRTS-1-VEGF-A construct
rTetR Reverse tetracycline repressor
rtTA Reverse tetracycline-controlled transactivator
RUNX2 (also known as Cbfa-1) Runt-related transcription factor 2
SP7 (also known as Osx) Osterix
Tc or Tet Tetracycline
Tet-Off Tetracycline-based gene repressor system
Tet-On Tetracycline-based gene inducible system
TetO Tetracycline Operator
TetR Tetracycline Repressor
TetRKRAB
Tetracycline Repressor (Kruppel-associated box)
TRE Tetracycline Response Element
tTA Tetracycline-controlled Transactivator
tTS Tetracycline Transcriptional Silencer
tTSKRAB
Tetracycline Transcriptional Silencer modified with Kruppel-associated box
UCB Umbilical Cord Blood
USSC Unrestricted Somatic Stem Cells
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List of Tables
Table
Number
Full Description Page
3.1.1.1 Restriction Enzyme Diagnostic Digest Protocol 34
3.1.1.2 Enzymatic Activity Temperature Guideline for Restriction Enzymes 34
3.1.1.3 Restriction Enzyme Digestion Protocol 36
3.1.2.1 PCR Gene Amplification Protocol 38
3.1.2.2 PCR Amplification Program – KPTOUCH Protocol 38
3.1.3 Ligation Protocol 39
3.1.4 DH5α Transformation Protocol 39
3.1.5 pRTS-1-X Diagnostic Digest – Orientation Confirmation of Gene Insert 41
3.2.1 Cell Thawing Protocol 42
3.2.2 Trypan Blue Cell Viability Protocol 42
3.4 Hygromycin B Concentration Levels Use for Kill Curve Experiment 43
3.5.3 SuperFect Optimization Experiment (DNA and DNA:SuperFect Ratio) 47
4.4.1 Summary of Nucleofection Experiments 65
4.4.5.1 Bio-Rad Electroporation Voltage Gradient Results 70
4.4.5.2 Bio-Rad Electroporation Duration Gradient Results 70
5.1 Summary of All Transfection Experiments in Chronological Order 84
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List of Figures
Figure
Number
Full Description Page
1.2 Venn diagram of Tissue Engineering Triad 8
1.3.3.1 Cartoon depiction of the Tet-Off system 12
1.3.3.2 Cartoon depiction of the Tet-On system 12
1.3.4.1 Cartoon depiction of the enhanced Tet-On system (Silencer) 14
1.3.4.2 Cartoon depiction of the fully enhanced Tet-On system found in the pRTS-1
Plasmid
15
1.3.4.3 Schematic map of the pRTS-1 plasmid 16
1.3.5 HUCPVC and the SEM of an excised umbilical artery indicating the location
of HUCPVCs
17
3.1 Schematic map of the pRTS-1 plasmid 33
3.1.2 Primer details of gene inserts 37
3.5.5 Diagram of the V-grad/D-grad Program 48
4.1.1 Sfi I Restriction Sequence and its Unique 5‟ Overhang 54
4.1.2 Confirmation of Insert Orientation through Diagnostic Digest 55
4.2 HUCPVC Characterization 56
4.3.1 HUCPVC Kill Curve 56
4.3.2 RBMC Kill Curve 57
4.3.3 ROS Kill Curve 57
4.4.1.1 Nucleofection Optimization Kit Solution L and V Transfection (24 Hours) 59
4.4.1.2 Cell viability after four days of selection 60
4.4.1.3 Nucleofection Optimization Kit Solution L and V GFP expression (X-001) 61
4.4.1.4 Diminishing GFP expression of Nucleofection transfected HUCPVCs 61
4.4.1.5[A] MSC Kit Nucleofection (A-020) 62
4.4.1.5[B] MSC Kit Nucleofection (C-017) 63
4.4.1.5[C] MSC Kit Nucleofection (P-016) 63
4.4.1.5[D] MSC Kit Nucleofection (X-001) 64
4.4.2 Lipofectamine 2000 transfection (HUCPVC) 66
4.4.3.1 Qiagen SuperFect transfection (HUCPVC) 67
4.4.3.2 Qiagen SuperFect transfection (RBMC) 67
4.4.4.1 Clontech Xfect transfection (HUCPVC, RBMC, ROS Cell) 68
4.4.4.2 Long Term decrease in GFP expression (RBMC, ROS Cell) 68
4.4.4.3 Formation of large complexes and cell disintegration caused by confluence 69
4.4.4.4 Xfect Transfection with 50% Confluence 70
4.4.5.1 Bio-Rad Electroporated RBMCs and ROS Cells express GFP after 12 hours 71
4.4.5.2 Bio-Rad Electroporation (DNA optimization) 71
4.5.1 Single Clone Expansion of ROS-pRTS-1-BMP-2 after 13 days 72
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4.5.2 Xfect Transfected and Electroporated RBMC - Single Clone Expansion 73
4.7 mRNA Expression of all genes from transfected RBMC and ROS cells 74
4.8 Western Blot of all transfected RBMC and ROS cells 75
4.9 Doxycycline Dose-Response after 24 Hours (RBMC and ROS Cell) 76
4.10.1 Induction Kinetics of RBMC-RUNX2 and ROS-RUNX2 78
4.10.2 pRTS-1 Transfected RBMC Doxcycline Kinetics 79
4.11.1 ALP activity Assay (Conditioned Media) 80
4.12.2 ALP activity Assay (Co-Culture) 81
5.5 Vector map of pRTS-1 highlighting the region with the Tet-On enhancement 93
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Chapter 1 – Introduction
Human bone is a specialized connective tissue with major functions that include: being a
primary mechanical support for locomotion, a physical barrier for vital organ protection, and a
reservoir for ions, particularly calcium, and growth factors.32, 74
However, like all tissues in the
body, injury or disruption to the bone tissue can occur and depending on the severity of the latter;
smaller injuries may be repaired through active remodeling while more extensive injuries may
require therapeutic intervention to regenerate bone.
Bone tissue comprises a mineralized extracellular matrix (ECM), which consists of both
inorganic and organic components. The main inorganic component is the hydroxyapatite (HA)
while the main organic component is type I collagen. Synthesized by osteoblasts, type I collagen
is composed of two αI chains and one αII chain (transcribed from Col1a1 and Col1a2 genes,
respectively) in a coiled coil structural motif. This structure undergoes fibrillogenesis and the
rate of deposition results in either woven or lamellar bone formation.84
In the human adult both trabecular, or cancellous, and cortical, or compact, bone are
lamellar in structure, although the former defines interstices for marrow and possesses a large
surface area for increased endocrine function, while the latter is dense and packed with osteons,
remodels more slowly than cancellous bone, but provides resistance to torsional loading during
locomotion. 32
Arising from both mesoderm and neural crest, bone is formed through a process known
as osteogenesis, and constantly remodeled through the individual cellular activities of osteoblasts
and osteoclasts.51
Osteoblasts are derived from mesenchymal/ectomesenchymal cells and
synthesize type I collagen as well as other specialized bone-related proteins such as bone
sialoprotein, osteocalcin, osteonectin, and osteopontin; and mineralize this organic component
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with hydroxyapatite (HA) microcrystallites. On the contrary, osteoclasts are multinucleated cells
that are derived from hematopoietic stem cells and function to resorb bone and maintain bone
homeostasis and active remodeling.
Although the molecular pathways resulting in osteogenesis have yet to be fully
understood, some proteins have been shown to be critically necessary for osteoblastic
differentiation. One major group of proteins is the Bone Morphogenetic Protein (BMP) family
which belongs to the TGF-β superfamily.10
In the 1960s, Urist et al. discovered auto-inductive
osteogenic activity when a decalcified bone matrix was placed into the belly of the rectus
abdominus muscle of a rabbit.114
This transplant resulted in ectopic bone formation and the
finding quickly became the basis of the field of bone induction research. However, is was not
until the 1980 that Wozney et al.126
first cloned and sequenced BMP, since which time BMPs
have been found to have multiple functions in embryonic development, bone induction, and
other growth and differentiation cascades, although this thesis will focus on the impact of BMP-
2 on bone induction.10
In fact, in this thesis, three osteoinductive genes and an angiogenic gene were chosen as
targets to be selectively expressed in a variety of cell types, to assess their potential effects on
bone induction for the purpose of tissue regeneration. The three osteoinductive genes were BMP-
2, Osterix (Osx or SP7), and Runt-related transcription factor 2 (RUNX2 or Cbfa-1). BMP-2, as
mentioned above, is a powerful cytokine selected for its ability to promote osteoblastic
differentiation.10
OSX has been reported to be an inhibitor of chondrogenesis75
, while an OSX
knockout mouse revealed complete absence of osteoblasts, which is indicative of the critical
importance of OSX in osteoblastogenesis.75
The third osteoinductive gene, RUNX2, has been
found to be up-regulated in hypertrophic chondrocytes and is another crucial gene for
osteoblastogenesis.37
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Current literature suggests a complex signaling pathway associated with these three genes.
For example, the expression of both OSX and RUNX2 are up-regulated by BMP-2 through the
SMAD pathway.37
Early studies indicate OSX as a downstream gene of RUNX2 due to the lack
of OSX expression in RUNX2-/-
mice.24
Overexpression of RUNX2 induced expression of OSX
in MSCs, thus, the expression of OSX was controlled by BMP2 via RUNX2.75
However, it has
been recently demonstrated that transfection of OSX into RUNX2-/-
MSCs induced
osteoblastogenic activity.75
Furthermore, embryos deficient in either OSX or RUNX2 resulted in
the absence of bone8,24
but the phenotypes of the two null mice are different at birth, suggesting a
difference in function during bone formation.75
OSX deficient mice exhibited normal
chondrocyte differentiation while RUNX2 deficient mice presented neither osteoblasts nor
hypertrophic chondrocytes.24
Moreover, OSX and RUNX2 stimulate the expression of distinct
genes and thus, collectively, this suggests OSX functions as a downstream target of RUNX2 but
also functions independently of RUNX2 for osteoblastogenesis.75
Although the complete
signaling pathways are unclear, these three genes were selected for their involvement in
osteoblastogenesis.
The final gene selected for this thesis was the vascular endothelial growth factor A
(VEGF-A). All living tissues in the body are composed of cells and ECM. Cells require fresh
nutrients, oxygen, as well as a method of removing waste in order to function. Bone tissue is no
exception. Although the initial signal for the invasion of blood vessels into bone is not fully
known, it is believed that hypoxia triggers the signaling cascade.122
Hypoxic conditions trigger
cells to release hypoxia-inducible factor (HIF) and, as a result, activates VEGF, which in turn
promotes angiogenesis. Experimental blockade of angiogenesis resulted in decreased bone
density and disturbed bone mineralization.56, 122
Consequently, VEGF-A was included as part of
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this research study to induce angiogenesis and potentially improve the gene therapy treatments of
large bone defects.
1.1 – Non-Union Bone Defects and their Treatment
Bone fractures may be generalized into two categories, union and non-union defects. Due
to active matrix remodeling, fractures normally heal or are said to have achieved union, but
complications including excessive movement could increase the complexity of the injury. In
these cases, the damage or defect to the bone is too severe or too large for the natural restoration
process to take place and thus, a non-union defect is generated. Current treatments for replacing
bone through bone grafting or cytokine delivery of superphysiological doses (1.7-3.4mg) of
rhBMP-2125
require multiple invasive surgeries, which translates into further increase in the
overall cost of non-union bone treatments.
Bone grafts are separated into three major categories: autografts, allografts or xenografts,
and biomaterials. In autografts, bone is harvested from one site of the patient and transferred to
the recipient site. This procedure is most successful because it avoids immunological issues and
provides the cells and bioactive molecules to induce and sustain the regenerative process. A
major drawback of autografts is the limited availability of bone in the patient.
In contrast, the availability of allograft and xenograft bone is greater. In allografts, bone
is transferred from a cadaveric donor to the recipient patient. Allografts are common but there is
a non-zero risk of immune response and also the possibility of pathogen transfer, both of which
detract from this approach. Xenograft utilizes bone from an animal and is used rarely, and only,
as an end-stage organ failure treatment where a lack of alternative treatment will compromise the
life of the patient. This procedure has similar complications to allografts with the addition of
disease transmission, non-matching biological molecules as well as a different lifespan of the
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bone.124
In addition, graft tissues can first be demineralized to create a demineralized bone
matrix (DBM). DBMs are osteoconductive, can re-mineralize over a long period of time, but do
not provide mechanical strength for structural support until neo-osteogenesis has occurred.91
Finally, synthetic variants or biomaterials have been developed as an alternative source
for bone regeneration. These materials, such as hydroxyapatite and beta-tricalcium phosphates,
act as osteoconductive scaffolds for bone regeneration and can be supplemented with
osteoinductive agents such as BMPs, bone marrow aspirates53
, and cultured osteoblasts.11
For the
purpose of this thesis, osteoinductive refers to the ability to stimulate osteoprogenitor cells to
differentitate into osteoblasts to form new bone and osteoconductive refers to the growth of bone
on a surface. The two major benefits of this treatment are availability and biocompatibility, while
they are limited by not being osteoinductive (although some high surface area calcium
phosphates would appear to have osteoinductive properties, probably through the adsorption of
growth factors from the host blood stream64
); and they also exhibit slow degradation and high
radiodensity.53
In addition to the grafts and biomaterials briefly reviewed above, increasing the blood
supply to the bony wound site, can enhance the regenerative process. In 2004, Munk and Larsen
conducted a systematic review of 147 publications that included 5246 cases of scaphoid non-
unions. Their study included literature from 1928 to 2003 and focused on non-vascularized bone
grafting with or without internal fixation and vascularized bone grafting outcomes. The
consensus reached was that a vascularized regeneration process results in a significant increase
in the rate of union for bone grafting compared to non-vascularized processes.80
Other experimental techniques such as Guided Tissue Regeneration (GTR) and Guided
Bone Regeneration (GBR) have been explored with limited successs.34,95
Introduced in the 1980s,
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the Guided Tissue Regeneration (GTR) principle states that the regeneration of a specific tissue
is achieved when the cells with the regenerative capacity are allowed to populate during
regeneration.95
In 1988, the Guided Bone Regeneration (GBR) protocol was developed and in
this treatment, the surface of the bone is coated with a cell-occlusive membrane that prevents soft
tissue invasion while permitting osteoprogenitor cells to be recruited to increase the overall
osteogenic activity.95
1.2 – Tissue Engineering Triad
The aim of tissue engineering is to generate new tissue which restores both structure and
function to the site of injury without adverse effects. To generate this tissue, three issues should
be considered: cells, cell signaling factors, and scaffolds.
First, the cells must be evaluated for their availability and immunophenotype. Specific
cell types are better suited for targeted environments and stem cells are often selected for their
capacity to differentiate into multiple lineages.95,101
One issue with stem cells is their availability;
in a study conducted by Horwitz et al., six patients with severe osteogenesis imperfecta received
two infusions of bone marrow-derived mesenchymal progenitor cells at a median dose of
4.68x106 cells/kg of body weight.
39 In order to obtain this large number of cells for each
therapeutic treatment, primary cells have to be expanded in vitro but the frequency of colony-
forming unit-fibroblast (CFU-F) of stem cells can vary greatly.102
For example, unrestricted
somatic stem cells (USSC) which are isolated from human umbilical cord blood (UCB)60
have a
CFU-F frequency of 1:200 million, and a cell yield only 4 times out of 10 attempts, while the
commonly used human bone marrow (BM) stem cells are found at a frequency of 1:10,000 at
birth, diminishing to 1:100,000 in adult marrow.102
More recently, an alternate source of
mesenchymal stem cells have been isolated from the umbilical cord tissue known as the Human
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Umbilical Cord Perivascular Cells (HUCPVC – described in more detail in Section 1.3.5). These
cells have been shown to have a CFU-F frequency of 1:333 at harvest that increases to 1:3 after
two passages.102
Thus, HUCPVCs represent an interesting putative source of stem cells for future
therapies. In addition to the availability, cells must either possess the immunophenotype of the
host, or be immunoprivilieged, to prevent undesired immune response. Clinically, mesenchymal
stem cells (MSC) have shown to be immunoprivileged thus avoiding this issue, and making
MSCs a preferred choice for tissue engineering. 40,102
The second condition of the tissue engineering triad (TET) is to provide the proper cell
signaling factor(s) at the site of implantation to induce tissue regeneration. This requires either
the exogenous addition of growth factors, or the employment of well-characterized genes to
propagate and enhance the regeneration process. To achieve the latter, a therapeutic gene is
genetically modified into plasmids and delivered into cells through replication-deficient
recombinant viruses or DNA molecules/complexes.125
The genetically engineered cells are then
selected for transgene expression, thus, allowing for in situ production of cell signaling factors.
In bone, the BMP-2 gene has been known to have a critical role in initiating osteogenesis114
and
thus was selected as one of the target genes for this thesis.
The final condition of the TET is the tissue scaffold. In some approaches, cells or
recombinant genes are transplanted using biopolymers or collagen gels.78,120
These artificial
carriers must be biocompatible and biodegradable for the new tissue to be formed; in addition,
the extracellular matrix of the region should be unaffected by the foreign or artificial material, to
minimize disturbance to tissue regeneration.
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Together, these requirements form the tissue regeneration triad and once all conditions
are satisfied, the newly generated tissue should restore the structure and function to the site of
injury.
1.2.1 – rhBMP-2 Treatment and their Limitations
The development and use of a recombinant human bone morphogenetic protein 2
(rhBMP-2) began in 1997 and in 2002, the rhBMP-2 was approved by the Food and Drug
Administration (FDA) for clinical interbody spinal fusion and to treat acute open tibial shaft
fractures.124
Since then, the application for this treatment was extended to oromaxillofacial
procedures, alveolar cleft repair, and a variety of orthopedic disorders.63
In clinical studies, the rhBMP-2 delivery utilizes superphysiological doses at orders of
magnitude greater (1.7-3.4mg) 125
than the serum physiological levels of pg/mL.86
Milligrams of
rhBMP-2 are absorbed in a collagen sponge, which is then placed into non-union bone defects.
Figure 1.2 Tissue Engineering Triad – Venn diagram depicting the three components and the necessary
combination required for a successful tissue regeneration (indicated by the yellow triangle).
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Clinically, numerous successful cases of bone regeneration have been reported with this
treatment but, more importantly, the success of this treatment addressed two major factors:
morbidity associated with transplantation complications and limited availability of bone tissue
associated with bone grafts.120
However, there are limitations to this treatment and its drawbacks
lie with the rhBMP-2 itself.12
The first limitation of rhBMP-2 is its short systemic half-life of 7 to 16 minutes6 and the
second limitation is the rate of release from the carrier. Pharmacokinetic release profile of
rhBMP-2 showed an initial, “burst”, release of more than 80% of rhBMP-2 within 10 minutes,
followed by a secondary long-term release of 1 to 10 days. The duration of the secondary release
phase is determined by the biomaterial carrier used for delivery.12
These two limitations result in
inefficient tissue regeneration and, thus, large quantities of rhBMP-2 are used for this
treatment.12
Furthermore, the risks and side effects of delivering superphysiological levels of rhBMP-
2 have not been fully studied; nonetheless, there are reports of increased osteoclastic resorption48
and areas of bone-void formation in long-term regeneration that are suspected to be caused by
the high dose of rhBMP-2.106
Furthermore, it has been suggested that superphysiological doses
of rhBMP-2 may “leak” and stimulate ectopic bone growth. In the cervical spine, this can cause
soft-tissue swelling that could lead to detrimental effects such as airway compromise.88
Other
complications such as the development of renal insufficiency have also been reported.63
One goal
of the current thesis was to explore an alternative delivery platform for BMP, and other
osteogenic-related genes, that would minimize such negative side effects associated with
supraphysiological dosing.
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1.3 – Methods to Control BMP2 Release
Different approaches have been reported in the literature that address issues associated
with the short half-life and burst release of rhBMP-2: genetically modifying rhBMP-2 and
inclusion of a cell system to express BMP-2 in vivo.
1.3.1 rhBMP-2-CBD
As discussed earlier, rhBMP-2 delivery is clinically used in conjunction with the only
FDA-approved carrier, collagen type I, to induce osteogenesis. One suggested method of
addressing the rapid release limitation is to prolong the release of rhBMP-2 from the collagen
carrier. To achieve this, Visser et al. genetically engineered rhBMP-2 to include a decapeptidic,
collagen type I binding domain (CBD) without disrupting its osteogenic function. This rhBMP-
2-CBD protein decreased the rate of efflux by binding to collagen fibres and, as a result,
prolonged the period of release by a week or longer.120
By extending the effective duration of this
protein, this modification could lower the quantity of rhBMP-2 used and subsequently reduce the
cost as well as the associated side effects. Although this modification addresses the burst release
of rhBMP-2 from the carrier, a direct delivery of the protein does not address the issue of half-
life as discussed in Section 1.2.1.
1.3.2 rhBMP-2-Cell System
As outlined above, successful tissue regeneration requires three conditions:
immunoprevileged cells expressing the correct cell signaling factors embedded in a
biocompatible scaffold. Thus, the second method reported in the literature to overcome the
problems of half-life and burst release is to genetically engineer cells to produce rhBMP-2 in
vivo. This modification could prolong the presence of rhBMP-2 in a non-union defect, thus
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11
potentially shortening the regeneration process through active protein production. However, this
approach presents an additional problem: that of needing to control the amount and duration of
rhBMP-2 expression. Without a control switch this approach could also ultimately result in
overproduction of rhBMP-2 and cause similar complications to those discussed above.
Such a control switch has, in fact, been described (see below) and employs tetracycline to
either switch on, or off, a gene of interest and thus tightly regulate the expression level and the
duration of gene therapy.
1.3.3 The Control Mechanism: The Tet-Off and Tet-On Systems
In the past twenty years, experimental tools have been developed and enhanced to allow
conditional modulation of gene activity in eukaryotes with high specificity.29
One breakthrough
was the development of the Tet-On (i.e., tetracycline-on) system.29
This system is a modification
of the Tet repressor (TetR), tetracycline (Tc or Tet), and tetracycline operator (TetO) interaction
used in the Tet-Off system.
In the Tet-Off system, the Tc-controlled transactivator (tTA) protein is continuously
produced and functions differently in the presence or absence of Tc. In the absence of
doxycycline (dox), a member of the tetracycline antibiotic group, tTA binds to the TetO or the
Tet Response Element (TRE) located upstream of the promoter containing the gene of interest
and induces transcription. On the other hand, in the presence of dox, the tTA binds to dox and is
prevented from binding to the TetO. Thus, transcription of the gene of interest stops, as seen in
figure 1.3.3.1.29
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The Tet-On system utilizes the same TetR-Tc-TetO interaction but with a modified,
reversed effect in the presence or absence of doxycycline. Gossen et al. randomly mutagenized
the TetR gene and screened for Tc dependence of repression in vivo. They found one mutant
with four amino acid changes (Glu71 Lys
71, Asp
9 5 Asn
95, Leu
101 Ser
101, Gly
102 Asp
102)
that produced a reverse phenotype and increased expression of the gene of interest 30-fold.30
This mutated TetR was renamed to reverseTetR (rTetR) for its opposite effects to presence or
absence of doxycycline and subsequently the tTA was renamed to rtTA.
Figure 1.3.3.2 The Tet-On System – The gene of interest is only transcribed in the presence of doxycycline.
Figure 1.3.3.1 The Tet-Off System – The gene of interest is only transcribed in the absence of doxycycline.29
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As visualized in figure 1.3.3.2, in the presence of doxycycline, rtTA binds to TetO and
the gene of interest is expressed at a dose-dependent level.30
1.3.4 pRTS-1: The Enhanced Tet-On System
Constructed by Bornkamm et al., the pRTS-1 plasmid is an Epstein-Barr Virus (EBV)-
derived episomally replicating plasmid that includes all elements of the inducible gene
expression system.4 This one plasmid system advances the traditional Tet-On system by
improving the ease of use, suppressing the background activity, and simultaneously expressing
two genes.
The traditional Tet-On system consists of two plasmids: a regulator plasmid and a
response plasmid. The regulator plasmid contains the elements to produce the rtTA protein while
the response plasmid contains the rtTA binding element as well as the inducible gene of interest.
It has been reported that with the traditional system, the best results are obtained when the two
plasmids are transfected in a two-step procedure.4 In the first step, transfected cell lines with
proper rtTA expression are selected, followed by a secondary transfection and selection steps
with the response plasmid. In the pRTS-1 system, Bornkamm et al. joined the regulator and the
response plasmid into one. This reduces the need for multiple transfections, and it has been
suggested that this modification can be of great advantage for difficult transfections.
Furthermore, by reducing the number of steps, the time required to obtain the final cell product is
reduced, which is crucial for primary cells.
The second improvement to the traditional Tet-On system is the addition of a
transcriptional silencer, tTS. Normally, episomally replicating plasmids avoid the influence of
chromosomal integration; however, integration in appropriate chromosomal loci is possible.4 As
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14
a result, significant rtTA-independent activity may be observed, but the addition of a tTS
prevents unspecific “leaky” expression by shielding TetO (refer to figure 1.3.4.1).
Furthermore, rtTA is replaced with point mutated rtTA2S-M2 (Asp
148 Glu
148 and His
179
Arg179
), which exhibits increased dox sensitivity with lower background activity116
, and the
tTS is replaced with the modified tTSKRAB
(Fusion of the repressor protein to a strong
transcriptional repressor, Kruppel-associated box “KRAB”), which binds downstream of the
TATA box to also lower the background activity.23
Simultaneous expressions of both regulators
create a stringent doxycycline-dose response system and it was reported that the background
activity was decreased by 1-2 orders of magnitude. As a result of this low background activity,
induction ranged from 1000- to 140, 000-fold whereas a traditional Tet-On system ranged from
30- to 100-fold increase.4
Figure 1.3.4.1 Enhanced Tet-On System (Silencer) – In the absence of doxycycline, tetracycline-dependent
transcriptional silencer (tTS) has a higher binding affinity to TetO and prevents unspecific expression (Left). In
the presence of dox, both tTS and rtTA bind with dox and reverse the binding affinity to TetO, resulting in gene
expression (Right).
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15
The final enhancement to the traditional Tet-On system is found in the response element.
The regulator element of the pRTS-1 plasmid consists of a bicistronic expression cassette that is
driven by a chicken-beta-actin promoter, which is active in many eukaryotic cell types.4 The
pRTS-1plasmid contains a bidirectional promoter, PTetbi-1, which expresses two genes
simultaneously and at equivalent levels (refer to figure 1.3.5.2).36
The secondary position of the
bidirectional promoter may encode a secondary gene of interest or a reporter gene such as eGFP
to rapidly monitor expression of the gene of interest.4
Figure 1.3.4.2 pRTS-1: Enhanced Tet-On System – In the absence of doxycycline, tetracycline-
dependent transcriptional silencer (tTSKRAB
) has a higher binding affinity to the bidirectional promoter and
prevents unspecific expression of both the gene of interest and the reporter gene (Left). Conversely, in the
presence of dox, both tTSKRAB
and rtTA2s-M2 binds with dox and reverses the binding affinity to the
bidirectional promoter, resulting in 1000- to 140 000-fold increased gene expression (Right)
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This pRTS-1 plasmid, developed by Bornkamm et al., is a technological improvement
that allows for a tightly regulated, doxycycline dose-dependent expression of the gene of interest,
and it was genetically modified to express the genes BMP-2, RUNX2, OSX, VEGF-A in this
thesis.
1.3.5 The Cells
Since the development of cell therapy treatment, bone marrow-derived mesenchymal
stem cells have been a common source of cells for cell-based therapy.125
The mesenchymal
population of the BM have the capacity to differentiate into a wide range of tissues including
tissues of the musculoskeletal systems.102
Thus, by genetically engineering cells with the
capacity to differentiate, the system aims to induce an autocrine and paracrine osteogenic
signaling at the site of implantation. According to the U.S. National Institute of Health
(www.clinicaltrials.gov), there are 165 clinical trials conducted with mesenchymal stem cells, of
which 100 studies are currently on-going.
Figure 1.3.4.3 Schematic map of pRTS-14 – Schematic map of the pRTS-1 plasmid. This system enhances the
traditional Tet-On system by incorporating both the regulator and response element into a single plasmid,
implementing a silencer and modifying the rtTA, and modifying the response element with a bidirectional
promoter.
Page 28
17
For the purpose of this research thesis, three cell types were transfected to develop the
tightly regulated inducible system: Human Umbilical Cord Perivascular Cells (HUCPVC), Rat
Bone Marrow Cells (RBMC), and Rat Osteosarcoma cells (ROS). Cells were selected based on
their potential use as well as their abilities to overcome difficulties in transfections encountered
throughout this research.
HUCPVC is a mesenchymal stem cell derived from the umbilical cord. During the
growth of a fetus, the human umbilical cord reaches 48-60cm in only 40 weeks19
and it has been
postulated by Davies et al. that this rapid growth must be accompanied by rapidly proliferating
stem cells.102
These multipotent cells are found adjacent to the vasculature of the umbilical cord
and give rise to the connective tissue of Wharton‟s Jelly.102
To date, HUCPVC studies indicate
that they are similar to BM-MSC in terms of differentiation, gene expression, and profiling
markers. With their ability to evade allogeneic host immune recognition, these cells would be
optimal for tissue engineering treatments.19
Figure 1.3.5 Human Umbilical Cord Perivascular Cells and its location present in the umbilical cord102
– (A) Scanning electron microscopy of the umbilical artery excised from the umbilical cord. The dotted line
depicts the outer margin of the perivascular region where HUCPVCs are found. (B) HUCPVCs in vitro
presenting a fibroblastic morphology.102
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In addition to HUCPVCs, Rat Bone Marrow Cells (RBMC) and Rat Osteosarcoma cells
(ROS) were employed herein to develop the pRTS-1-based controlled expression system as the
current state of HUCPVC characterization is not complete and it has been shown that primary
human cells are more refractory to transfections. In anticipation of such difficulties, RBMC and
ROS cells were transfected as alternative cell lines to overcome two major issues: transfection
difficulties and diminished proliferation post-transfection. Detailed discussions of these two
issues are found in Chapter 5. Original RBMC were obtained from the femoral bones of young
adult (120-130g) male Wistar rats. From those, non-transformed clonally derived RBMCs were
selected. RBMCs D8 (Materials # 46) are spontaneously immortalized stable population of cells
that upregulates RUNX2, OSX expression. Furthermore, these cells generate bone-like nodules
in the shortest time reported to date in supplemented culture media.50
Unlike the HUCPVC, these
cells are not immunoprevileged but these primary bone marrow cells have the capacity to
differentiate into osteoblasts under the correct conditions and begin osteogenesis.107
ROS,
specifically subclone ROS 17/2.8 was derived from a spontaneous tumor in an ACI rat by
Majeska et al. in 1980.71
This immortalized osteosarcoma cell line constitutively express
osteocalcin93
, and has increased alkaline phosphatase activity and type I collagen production.71
1.4 Transfection Methods
Transfection, which describes the process of incorporating an external gene into a cell,
can be broadly separated into three categories, chemical, electrical, and viral transfections.
Different transfection methods accomplish gene incorporation differently but the end results of
transfections must meet several criteria. First, cellular disruption such as membrane destruction
must not occur. Secondly, cell death post-transfection must be minimal and lastly, the
transfection efficiency must be high.
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19
Viral transfections are a widely used method of transfection for gene therapy. There are
five main groups of viral vectors currently employed: adenovirus, lentivirus, retrovirus, adeno-
associated viruses (AAV), and herpes simplex virus-1 (HSV-1).111,118
Viral transfections have
shown success in implementing genes into the genome but the drawbacks such as insertional
mutagenesis and immune response in patients makes viral transfections less desirable. Thus,
alternative methods of gene delivery have been studied.
Amaxa Nucleofector II
The Amaxa nucleofector II is an electrical-based transfection device where the negatively
charged DNA is driven into the cells through a unique proprietary cell-type-specific electrical
parameter. Nucleofection was developed to allow efficient gene delivery directly into the nucleus
of the cell, thus, has been described as an efficient transfection method for primary cells.31,33
For
this research, two kits were used for nucleofection: Cell Line Optimization Nucleofector Kit
(Materials #76) and Human MSC Nucleofector Kit (Materials #78). It is important to note that
both kits require a unique buffer with proprietary composition that negatively affects cells during
prolonged exposure.
Invitrogen Lipofectamine 2000
Invitrogen Lipofectamine 2000 is a chemical transfection method that utilizes cationic
lipid molecules that bind to the negatively charged DNA to form a DNA-Cationic lipid complex.
This complex is then fused with the cell and the DNA is released to complete a successful
transfection. Zhao et al. demonstrated successful transfection to deliver siRNA into human
embryonic stem cells with high efficiency.133
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Qiagen SuperFect
Qiagen SuperFect is a chemical transfection method whereby positively charged
nanoparticles interact with the negatively charged DNA to form a toroid-like structure. This
complex then binds to the surface of the cell for a nonspecific endocytosis.
Clontech Xfect
Clontech Xfect is a chemical transfection method that uses a biodegradable transfection
polymer that forms a nanoparticle complex with the DNA. Xfect has been developed with low
cytotoxicity to minimize cell death during transfection and this allows for higher quantities of
DNA complexes to be applied to each transfection, thus allowing for higher transfection
efficiency.
Bio-Rad Gene Pulser MXcell Electroporation System
Bio-Rad‟s Gene Pulser MXcell Electroporation System is an electrical-based transfection
system whereby the individual parameters of an electroporation can be modified, specifically the
following: time constant, voltage applied, pulse interval, pulse duration, and pulse type (square
or exponential). These parameters allow the system to be optimized for primary cells.
Furthermore, unlike the nucleofector, transfection for the Gene Pulser is completed in Opti-
MEM (Materials #52) which removes the cytotoxic effects associated with the proprietary
buffers of the nucleofector (Amaxa). This allows the user to optimize the transfection parameters.
1.5 Rationale
The current delivery of BMP-2 at super-physiological doses have achieved clinical
success in bone regeneration but a wasteful burst release of this expensive osteoinductive protein
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warrants a need for effective method of delivering osteoinductive proteins. By genetically
engineering mesenchymal cells with osteogenic genes and regulating the expression, a
continuous presence of BMP-2 at the site of regeneration should be possible. This ability to
control the quantity as well as the duration of osteoinductive genes present during bone
regeneration should quicken the regenerative process and remove the need for multiple
expensive injections of BMP-2.
1.6 Hypothesis
It is hypothesized that:
1. Stably transfected pRTS-1 mesenchymal cells will continuously express the gene of interest
upon exposure to doxycycline
2. The dose and duration of gene expression can be tightly regulated by the concentration and
exposure length of doxycycline
1.7 Objectives
The specific objectives of this work are:
1. To generate stable clones of HUCPVCs, RBMCs, and ROS cells transfected with each of the pRTS-
1-X plasmids: optimal method of transfecting the large 18+kbp plasmid will be investigated.
2. To investigate the expression pattern of pRTS-1-X transfected cells: doxycycline dose-dependent
expression and the induction kinetics of the genes of interest will be investigated.
3. To investigate the osteogenic potentials of overexpressing BMP-2, RUNX2, and OSX:
overexpressing the necessary genes for osteogenesis and their effects on osteogenesis will be
investigated.
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Chapter 2 – Materials
This chapter lists all chemical, cell culture reagents, and additional materials used in my
experiments. They are organized into corresponding experiments with complete details of origin.
# Materials Vendor Catalog Number
Additional Information
2.1 General Materials
1 Round-Bottom Tubes (14.0 ml)
BD Falcon 352057
2 Conical Tubes (15
ml) BD Falcon 352095
3 Conical Tubes
(50 ml) BD Falcon 352070
4 Cell Scraper, 25cm
(1.7cm blade) Sarstedt 83.1830
5 Glass Pasteur
Pipette 9" Fisherbrand
2559 - 136786H
6 Serological pipette
(5ml, 10ml, 25ml) Sarstedt
86.1253.001 86.1254.001 86.1685.001
7 Microtubes
(1.5 ml) Axygen 311-08-051
8 PCR Strip Tubes
(0.2 ml) VWR 53509-304
2.2 Cloning Materials for pRTS-1-X Constructs
Chemicals & Reagents
9 Agar BioShop AGR 001.1
10 Agarose BioShop AGA 001.500
1% Agarose Gel:
- 2mL of TAE buffer
- 98mL of dH2O
- 1g of Agarose
- EtBr Concentration 10 mg/mL
11 Ampicillin BioShop AMP 201 1000x Concentration
of 50 mg/mL
12 Competent DH5α
Cells Invitrogen 18265-017
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23
13 DNA Ladder
(100bp)
New England Biolabs
N3231S
14 DNA Ladder
(1kbp)
New England Biolabs
N3232S
15 DNA Ladder
(1kbp Plus) Invitrogen 10787018
16 EDTA BioShop EDT 001.500
EDTA Solution Composition (1L) :
- 186.1g of EDTA in 700mL H2O
- Adjust pH to 8.0 with 10M NaOH
- Add H2O to 1 L
17 Ethidium Bromide BioShop ETB 333.1
18 Gel Loading Dye
(6x) New England
Biolabs B70221S
19 Glycerol BioShop GLY 002.1
Glycerol Solution Composition (per 100ml) :
- 65% Glycerol (v/v)
- 0.1M MgSO4
- 0.025M Tris pH 8.0
20 Kanamycin Sulfate BioShop KAN 201.5 1000x Concentration
of 30 mg/mL
21 LB Broth Lennox BioShop LBL 405.1
22 Platinum Taq DNA
Polymerase Invitrogen 10966018
100rxns
23 T4 DNA Ligase New England
Biolabs M0202S
24
TAE (Tris/Acetate EDTA)
Electrophoresis buffer
TAE Electrophoresis Buffer Composition (50x Stock Solution) :
- 242g Tris base
- 57.1ml acetic acid
- 37.2g EDTA
- Add dH2O to 1 L
- pH 8.0
25 Tris BioShop TRS 001.1
26a Restriction Enzyme
(Bgl II)
New England Biolabs
R0144S
Page 35
24
26b Restriction Enzyme
(Hind III)
New England Biolabs
R0104S
26c Restriction Enzyme
(Not I)
New England Biolabs
R0189S
26d Restriction Enzyme
(Sfi-I)
New England Biolabs
R0123S
27 QIAprep Spin
Miniprep Kit (250) Qiagen 27106
28 EndoFree Plasmid
Maxi Kit (10) Qiagen 12362
29 QIAquick Gel
Extraction Kit (50) Qiagen 28704
Plasmids
30 pRTS-1-Luciferase pRTS-1 Base
Plasmid
Received from Dr. Georg W. Bornkamm at GSF-Institut fur Kilnische Molekularbiologie und Tumorgenetik, Marchioninistrasse 25, D-81377
Munchen, Germany4
31 pRTS-1-BMP2
plasmid Described in section 3.1
32 RUNX2 Human
plasmid Origene SC302270
33 SP7 Human
plasmid Open
Biosystems 8069055
Accession #BC065522
34 VEGF-A Human
plasmid Open
Biosystems 6006890
Accession #BC101549
35 Primers ACGT Corp. www.acgtcorp.com
Apparatus
36 Sequencing TCAG
Sequencing Server
http://tcag-sequencing.ccb.sickkids.ca/
37 PCR
Eppendorf 97017 Mastercycler gradient
Perkin Elmer GeneAmp PCR System 2400
38 Gel Doc XR
System Bio-Rad 170-8170
Page 36
25
39 Gel Electrophoresis Life
Technologies
Model H5 Series 1087
40 Max Q 5000
Shaking Incubator Thermo Scientific
SHKA5000
41 Nanodrop 1000 Thermo Scientific
Nanodrop 1000
42 Table Top Centrifuge
Eppendorf
5415D Non-refrigerated centrifuge for 1.5mL eppendorf tubes
5810R Refrigerated centrifuge for 15mL and 50mL Falcon tubes
2.3 Cell Culture
Cells
43
Human Umbilical Cord Perivascular
Cells
(HUCPVC)
Tissue Regeneration Therapeutics
www.verypowerfulbiology.com
Cord Numbers:
0408003, 0408004, 0508001, 0508002, 0508003,
44 Human Bone
Marrow Cells (hBM)
Tissue Regeneration Therapeutics
www.verypowerfulbiology.com
45 Rat Osteogenic Sarcoma Cells
(ROS) ATCC CRL-1663
www.atcc.org
46
Rat Bone-marrow derived
Mesenchymal stem Cells (RBMC),
clone D8
Generated in Dr. Sodek’s
lab
Reference # 50
General Reagents
47 Antibiotics (10X)
See Below for the
Individual Antibiotics
Antibiotics Composition:
- 2.5 µg/ml Fungizone
- 0.5mg/ml Gentamicin Sulphate
- 1650 units/ml Penicillin
48
Dulbecco’s Modified Eagle
Medium
(D-MEM) 1X
Gibco – Invitrogen
11995-065
Page 37
26
49
Minimum Essential Medium Alpha
(α-MEM) 1X
Gibco - Invitrogen
12561-056
50 α-MEM Freezing
Solution
α-MEM Freezing Solution Composition :
- 80% α-MEM
- 10% FBS
- 10% DMSO
51 D-MEM Freezing
Solution
D-MEM Freezing Solution Composition :
- 80% D-MEM
- 10% FBS
- 10% DMSO
52 Dimethyl Sulfoxide
(DMSO) Sigma-Aldrich 276855
53
Dulbecco’s Phosphate Buffered
Saline
(PBS) 1X
Gibco – Invitrogen
14190-144
54 Opti-MEM
Reduced-Serum Medium (1X)
Gibco – Invitrogen
11058-021
55 Fetal Bovine Serum Hyclone SH30397.03 Lot# K4M25240
and KPJ22093
56 Fungizone Gibco –
Invitrogen 15290-018
57 Gentamicin
Sulphate Gibco –
Invitrogen 15750-078
58 Parafilm University of
Toronto Medstore
2099-1337410 www.uoftmedstore.com
59 Penicillin G Sodium
Salt Sigma-Aldrich P3032-100MU
60 Trypan Blue Stain
0.4% Gibco –
Invitrogen 15250061
61 Trypsin Gibco –
Invitrogen 17072-018
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27
Plasticware
62
96-well Microplate, tissue-culture
treated, flat-bottom with lid
BD Falcon 353075
63
48-well Cell Culture Plates, tissue-culture treated
polystyrene, flat-bottom with lid
BD Falcon 353230
64
24-well Cell Culture Plates, tissue-culture treated
polystyrene, flat-bottom with lid
BD Falcon 353226
65
12-well Cell Culture Plates, tissue-culture treated
polystyrene, flat-bottom with lid
BD Falcon 353225
66
6-well Cell Culture Plates, tissue-culture treated
polystyrene, flat-bottom with lid
BD Falcon 353224
67 Dish 35X10mm TC BD Falcon 353001
68 Dish 60X15mm TC BD Falcon 353002
69 Dish 100x20mm TC BD Falcon 353003
70 Cell Culture Flask
(25 cm²) BD Falcon 353808
71 Cell Culture Flask
(75 cm²) BD Falcon 353136
Apparatus
72 Bright Field Microscope
Nikon 802162
73 Imaging Camera PixeLINK PL-A642
74 Cell Counter Beckman Coulter
Z1 Coulter Particle Counter
Page 39
28
75 Hemacytometer American
Optical
Bright line Hemacytometer
improved neubauer
0.1mm deep
76 Incubator Forma
Scientific
5% CO2, 37oC
2.4 Kill Curve
Additional Reagents
77 Hygromycin B Invitrogen 10687-010
2.5 Transfections
Additional Reagents
78 Cell Line
Optimization Nucleofector Kit
Amaxa-Lonza VCO-1001N Includes pmaxGFP control plasmid (3.4kbp)
79 Doxycycline,
Hydrochloride Calbiochem 324385
[200ng/ml] used unless specified otherwise
80 Human MSC
Nucleofector Kit Amaxa-Lonza VPE-1001
Includes pmaxGFP control plasmid (3.4kbp)
81 Lipofectamine 2000
Reagent Invitrogen 11668-027
82 SuperFect
Transfection Reagent (1.2 ml)
Qiagen 301305
83 Xfect Clontech 631317
Apparatus
84 Gene Pulser MXcell
Electroporation System
Bio-Rad 165-2670 96-well Plate Chamber :
165-2672
85 Fluorescence Microscope
Zeiss AXIOVERT
135M
86 Imaging Camera Hamamatsu C10600-10B
87 Nucleofector II Amaxa-Lonza
2.6 Transfections Efficiency Analyses
Apparatus
88 Fluorescence Activated Cell Sorter (FACS)
Beckman Coulter
Epics Altra
2.7 mRNA Expression Analyses
Additional Reagents
Page 40
29
89 CelLytic-M, Cell
Lysis Reagent, 250 ml
Sigma-Aldrich C2978-250ML
90 GenElute Direct
mRNA Miniprep kit Sigma-Aldrich DMN10
91
High Capacity RNA-to-cDNA
Master Mix with ‘No RT Control’, 50
rxns
Applied Biosystems
4390711
92
Protease Inhibitor Cocktail for use with mammalian cell and tissue extracts, 5 ml
Sigma-Aldrich P8340-5ML
Recommended Dosage:
100µl/ml at cell density
of 108 cells/ml
93 RNase-Free DNase
Set Qiagen 79254
2.8 Western Blot Analyses
Additional Reagents
94 Acrylamide/Bis-
Acrylamide (37.5:1) 30% Solution
BioShop ACR 010.500
95 Acrylamide/Bis-
Acrylamide (19:1) 40% Solution
BioShop ACR 003
96
Amersham ECL Advance Western Blotting Detection
Kit
GE Healthcare
RPN2135
97 Amicon Ultra
Centrifugal Filters – Ultracel – 30K
Millipore UFC803024
98 Ammonium Persulfate
Bioshop AMP 001.25
99 Anhydrous Ethyl
Alcohol
University of Toronto
Medstore
39752-PO16-EAAN
www.uoftmedstore.com
100
BioFlex MRI Single Emulsion X-Ray
Film 8 x 10" 100 sheets
CLMR810(for Radiology)
Clonex 2316-
CLMR810
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30
101 Bio-Rad Protein
Assay Bio-Rad 500-0006
102 DL-Dithiothreitol
(DTT) Caledon 27565-41-9
103 Ethyl Alcohol 95% University of
Toronto Medstore
39753-PO16-EA95
www.uoftmedstore.com
104 Immun-Star
Western C Kit Bio-Rad 170-5070
105 Isopropyl Alcohol EMD PX1835-5
106 Glycine BioShop GLN 001.1
107 Goat Anti-Mouse
IgG HRP Conjugate Bio-Rad 170-5047
Secondary AB used
at 1/25 000 dilution
108 Goat Anti-Rabbit IgG (H+L)-HRP
Conjugate Bio-Rad 170-6515
Secondary AB used
at 1/100 000 dilution
109 Immobilon – P Millipore IPVH00010
110 Methanol Caledon 67-56-1
111 PageRuler
Prestained Protein Ladder
Fermentas SM0671
112 Primary Antibodies Abcam
BMP2:ab17885
GFP:ab290
RUNX2:ab48811
SP7:ab57335
VEGF:ab9570
[BMP2]: 0.5 µg/ml
[GFP]: 1 µg/ml
[RUNX2]: 1.25 µg/ml
[SP7]: 2.5 µg/ml
[VEGF]: 10 µg/ml
113 SDS BioShop SDS 001.1
114 SDS
Electrophoresis Buffer (5x)
SDS Buffer Components :
- 15.1 g Tris base
- 72.0 g Glycine
- 5.0 g SDS
- H2O to 1 L
- pH 8.8
115 Skim Milk Powder BioShop SKI 400.500
116
N,N,N’N’ – Tetramethylethyl-
enediamine (TEMED)
BioShop TEM 001.25
Page 42
31
117
Tris-Buffered Saline-T
(TBS-T)
TBS-T Components :
- 100mM Tris-Cl, pH 7.5
- 0.9% (150mM) NaCl
- 0.1% Tween
- pH 7.4
118 Tween 20 BioShop TWN 510.500
Apparatus
119 ImageJ Software National
Institutes of Health
http://rsbweb.nih.gov/ij/index.html
120 PowerPac Universal
Bio-Rad 164-5097
121 Western Blot Cell Bio-Rad Mini-
PROTEAN II Tube Cell
122 Gel Transfer Apparatus
LKB 2197 Power
Supply 100mA per 0.75mm thick polyacrylamide gel
2.9 Alkaline Phosphatase Activity Assay
123 ALP solution
Suppliers of individual reagents
See below
ALP solution Components :
- 20 µl of 50mg/ml Naphthol AS-MX Phosphate dissolved in N,N - DMF
- 120 µl of 50mg/ml Fast Red TR or Fast Blue BB salt dissolved in dH2O
- 10 ml TM buffer (100mM Tris, pH 8.6, 10mM MgCl2)
124 Fast Blue BB Salt Sigma-Aldrich F-3378
125 Fast Red TR Sigma-Aldrich F-8764
126 Formaldehyde
Solution BDH UN1198
127 L-Ascorbic Acid Sigma-Aldrich A-5960
128 Magnesium
Chloride Sigma-Aldrich M9272-500G
129 Naphthol AS-MX
Phosphate Sigma-Aldrich N-4875
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130 N,N-
Dimethylformamide Sigma-Aldrich D4551-250ML
131 Paraformaldehyde Sigma-Aldrich P6148-1KG
132 rhBMP-2 Obtained
from Dr. Sean Peel’s lab
University of Toronto: Faculty of Dentistry
133 C2C12 Obtained
from Dr. Sean Peel’s lab
University of Toronto: Faculty of Dentistry
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Chapter 3 – Methods
Please refer to Chapter 2: Materials for details on all reagents and apparatus.
3.1 Cloning of pRTS-1-RUNX2/SP7/VEGF-A Constructs
The sub-cloning of the human genes of interest into the pRTS-1 plasmid (Materials #30)
was first designed. The pRTS-1-BMP2 plasmid was already available from Dr. Bernhard
Ganss‟s lab and was not created but used in this thesis.
The Tet-dependent bidirectional promoter, indicated by a red box in figure 3.1.1,
transcribes two genes simultaneously. For the purpose of this research, the portion containing the
luciferase gene was replaced with the gene of interest using the flanking Sfi-I restriction sites.
This exchange does not affect the doxycycline dose-dependent control mechanism and expressed
both the gene of interest and the eGFP reporter gene.
Figure 3.1 Plasmid map of pRTS-1 - Genes of interest were sub-cloned into the plasmid via the Sfi-I restriction
sites, indicated by arrows. The red box indicates the doxycycline-dependent bidirectional promoter.
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3.1.1 Preparation of pRTS-1 Plasmid Backbone
First, a sufficient amount of pRTS-1 plasmid was obtained. A stab culture of the glycerol
stock of pRTS-1-Luciferase plasmid (Materials #30) was placed in 3mL of LB broth culture
media (Materials #21) with 3µL of 1000x ampicillin (Materials #11) and incubated for
approximately 16 hours in a 37oC shaking incubator (Materials #40), set at 300 rpm. The LB
culture media (1 mL) was transferred into a 1.5mL eppendorf tube and centrifuged at 15 700 rcf
for 10 minutes in a tabletop centrifuge. The supernatant was carefully decanted from the
eppendorf tube and the replicated pRTS-1 plasmid was purified from the pelleted D5Hα cells
using the Qiagen Miniprep Kit (Materials #27). A Sfi I diagnostic digest was conducted on 5µL
of [5 µg/µL] eluted plasmid using the following protocol (Table 3.1.2.1):
Table 3.1.1.1 Restriction Enzyme Digest Protocol
Volume (µL) Item
5 µL DNA [5 µg/µL]
2 µL Restriction Enzyme Buffer Associated with Each Restriction Enzyme (10x
Concentrate)
2 µL BSA (Final Concentration 0.1mg/mL)
1 µL Restriction Enzyme (Table 3.1.2.2)
10 µL dH2O
20 µL Total Final Volume
Table 3.1.1.2 Enzymatic Activity Temperature Guideline for Restriction Enzymes
Restriction Enzyme Incubation Temperature (oC) Inactivation Temperature (
oC) Materials #
Bgl II 37oC N/A 26a
Hind III 37oC 65
oC 26b
Not I 37oC 65
oC 26c
Sfi I 50oC N/A 26d
Each restriction digests were conducted at the required incubation temperature in a PCR machine
(Materials #37) for 2 hours.
The digested and undigested (control) pRTS-1 plasmids were fractionated in a 1%
agarose gel (Materials #10) containing 50µg/mL of EtBr (Materials #17) and 5µL of 1kbp DNA
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ladder (Materials #14). Desired resolution was obtained through electrophoresis conducted in
TAE buffer (Materials #24) with 20µL of DNA and 5µL of loading dye (Materials #18). The gel
was then exposed to UV light and imaged in a gel dock (Materials #38).
Once the pRTS-1-Luc plasmid digest was confirmed, 3µL from the stab culture was
transferred into 100mL LB broth culture media containing 100µL of 1000x ampicillin (Materials
#11) and incubated for approximately 16 hours in a 37oC shaking incubator, set at 300 rpm. The
culture was then transferred into two 50mL falcon tubes (Materials #3) and centrifuged at 13 257
rcf for 30 minutes. The supernatant was carefully decanted and the replicated plasmids were
purified using the Qiagen Maxiprep Kit (Materials #28). The purified plasmids were eluted into
100 µL of elution buffer. The concentration and the quality of the plasmids were verified using a
Nanodrop (Materials #41).
Nanodrop 1000 Instructions
Prepare 100µL of dH2O, 50µL of DNA elution buffer, and 2µL of each construct. First,
run the application and set the scan for nucleic acids. Open the Nanodrop (Materials # 41)
apparatus and clean the loading area with dH2O and dry with Kimwipes. Load 1µL of dH2O and
begin calibration. Dry and load 1µL of the DNA elution buffer to calibrate the baseline. Clean
the loading area with dH2O and load the sample. Measure the quality and quantity of the DNA
using the provided software. Rinse with dH2O between each sample.
Lastly, to create the pRTS-1 plasmid without the luciferase gene, 45µL of the pRTS-1-
Luc plasmid was digested using Sfi I using the following digestion protocol (table 3.1.1.3).
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Table 3.1.1.3 Restriction Enzyme Digest Protocol
Volume (µL) Item
45 µL DNA [3 µg/µL]
6 µL Restriction Enzyme Buffer Associated with Each Restriction Enzyme (10x
Concentrate)
6 µL BSA (Final Concentration 0.1mg/mL)
3 µL Sfi I Restriction Enzyme (Table 3.1.2.2)
60 µL Total Final Volume
This digestion was conducted for 2 hours at 37oC and fractionated against an undigested pRTS-1
plasmid in a 1% agarose gel. The large band (approximately 17kbp) from the digested pRTS-1
lane was excised and purified using the Qiagen Gel Extraction Kit (Materials #29) and stored in
-20oC freezer until later use.
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3.1.2 Amplification of the Gene Inserts
The OSX, RUNX2, and VEGF-A gene inserts were created by PCR amplification using
custom made primers, designed specifically to amplify genes with flanking Sfi I restriction sites.
The primer design of each gene is as follows (figure 3.1.2):
Legend - Restriction Site (Sfi I)
- Start/Stop codon
- Gene of Interest
Name: VegP
VEGF-A
Forward Primer
5’-GTTC-GGCC-TCACT-GGCC-ATG-GCA-GAA-GGA-GGA-GGG-3’
Reverse Primer
5’-GTTC-GGCC-TCACT-GGCC-TCA-CCG-CCT-CGG-CTT-GTC-3’
Name: OsxP
Osx
Forward Primer
5’-GTTC-GGCC-TCACT-GGCC-ATG-GCG-TCC-TCC-CTG-CTT-3’
Reverse Primer
5’-GTCC-GGCC-TCACT-GGCC-TCA-GAT-CTC-CAG-CAA-GTT-3’
Name: RunP
RUNX2
Forward Primer
5’-GTTC-GGCC-TCACT-GGCC-ATG-GCA-TCA-AAC-AGC-CTC-3’
Reverse Primer
5’-GTTC-GGCC-TCACT-GGCC-TCA-ATA-TGG-TCG-CCA-AAC-3’
As described in figure 3.1.2, the forward primer contains the Sfi I restriction site attached
to the start codon for the genes of interest. The reverse primer contains the Sfi I restriction site as
well as the reverse complementary of the genes of interest. The designed primers were ordered
Figure 3.1.2 Primer Details of Gene Inserts - Primer details of genes to be amplified using the KP touch
protocol.
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from ACGT Corporation (www.acgtcorp.com). Lyophilized primers were hydrated with dH2O
(1µg/µL) and used in the PCR amplification protocol found in table 3.1.2.1 and 3.1.2.2:
Table 3.1.2.1 PCR Gene Amplification Protocol
Volume (µL) Item
2 µL ⁄ Diluted template plasmid including gene of interest
1 µL 0.1 µg/µL of forward primer
1 µL 0.1 µg/µL of reverse primer
2.5 µL 10x pfu buffer
2.5 µL 10mM dNTP mix
1 µL 50mM MgSO4
1 µL pfu Platinum Taq DNA Polymerase (Materials #22)
14 µL dH2O
25 µL Total Final Volume
Table 3.1.2.2 PCR Amplification Protocol - KPTOUCH Program
(Eppendorf Mastercycler Gradient PCR – Materials #37)
Step Number Temperature (oC) Duration (h:m:s)
1 94.0
0:04:00
2 94.0 0:00:30
3 60.0 0:00:30
4 72.0 0:00:30
5 94.0 0:00:30
6 59.0 0:00:30
7 72.0 0:00:30
8 94.0 0:00:30
9 58.0 0:00:30
10 72.0 0:00:30
11 94.0 0:00:30
12 57.0 0:00:30
13 72.0 0:00:30
14 94.0 0:00:30
15 56.0 0:00:30
16 72.0 0:00:30
17 94.0 0:00:30
18 55.0 0:00:30
19 72.0 0:00:30
20 Back to step 17 Repeat 35 times
21 72.0 0:10:00
22 4.0 Hold
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The amplified PCR products were digested with Sfi I using the digestion protocol in table
3.1.1.1. The digested products were fractionated on 1% agarose gel with 100bp and 1kbp DNA
ladders (Materials #13, 14). Gene inserts were excised and purified using the QIAquick Gel
Extraction Kit (Materials #29) and stored in -20oC freezer until further use.
3.1.3 Ligation of the pRTS-1-X Plasmids
The purified Sfi I digested empty pRTS-1 plasmid and the PCR-amplified inserts were
ligated using the protocol described below (Table 3.1.3) for 2 hours at 25oC:
Table 3.1.3 Ligation Protocol
Volume (µL) Item
1 µL Sfi I digested empty pRTS-1 plasmid
5 µL Sfi I digested gene insert
1 µL T4 DNA Ligase (Materials # 23)
2 µL T4 DNA Ligase Buffer
11 µL dH2O
25 µL Total Final Volume
3.1.4 Transformation of the pRTS-1-X Plasmids
10 µL of the respective ligation mixes were transformed into 50µL of competent DH5α
cells (Materials #12) using the following transformation protocol (Table 3.1.4).
Table 3.1.4 DH5α Transformation Protocol
Step Description
1 Mix 5µL of the ligated pRTS-1-X plasmid with 50µLof competent cells
2 Incubate on ice for 30 minutes
3 Heat shock for 20 seconds in a 42oC water bath without shaking
4 Incubate on ice for 2 minutes
5 Add 945µL of pre-warmed LB Broth culture media
The transformed cell suspensions were incubated for one hour in a shaking incubator
(Materials #40) at 37oC and 300 rpm. Cells were then centrifuged at 15 700 rcf for 5 minutes in a
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tabletop centrifuge. After 800µL of the supernatant was removed, the cells were resuspended in
the remaining 200 µL of supernatant by pipetting gently. The cell suspension was plated onto
prepared LB-Agar plates with 50µg/mL of ampicillin (Materials #11).
Ampicillin LB-Agar Plate Preparation
Mix 20g/L of LB broth (Materials #21) with 16g/L of Agar (Materials #9) and add dH2O
to the desired volume. Autoclave for 15 minutes then cool the bottle down below 55oC.
Ampicillin is heat sensitive and needs be added to the liquid LB-Agar mix below 55oC. Add
ampicillin and shake to mix. Distribute 15mL into 100mm culture dish and cool the plates until it
solidifies and store in 4oC fridge for future use.
Plates were incubated for 16 hours in a 37oC incubator (Materials #40) and 10 isolated
colonies were selected for screening. Stab cultures of the colonies were incubated for 16 hours
(37oC, 300 rpm) in 3mL of amp-added culture media. Plasmid isolation from each stab culture
was then conducted using the same method explained in section 3.1.2.
3.1.5 pRTS-1-X Orientation Diagnostic Digest
Due to the nature of Sfi I digests, gene inserts may be ligated in either direction into the
empty pRTS-1 plasmid, thus, diagnostic digest was performed on each pRTS-1-X construct to
confirm the orientation of the gene insert. The restriction enzymes (RE) were carefully selected
to cut once within the insert and once or twice in the plasmid. These DNA fragments were
fractionated using 2% agarose gel and the size of DNA fragments assessed. Table 3.1.5 presents
the RE used for each construct and their expected respective fragments released post-digestion:
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Table 3.1.5 pRTS-1-X Diagnostic Digest - Orientation Confirmation of Gene Insert
Construct Restriction
Enzyme Correct Orientation Incorrect Orientation
pRTS-1-RUNX2
Not I
(Materials #26c) 7500bp, 10 800bp 6400bp, 12 000bp
Hind III
(Materials #26b) 700bp, 5540bp, 12 000bp 800bp, 5430bp, 12 000bp
pRTS-1-OSX Bgl II
(Materials #26a) 2600bp, 15 600bp 1450bp, 16 750bp
After orientation was confirmed, the quality and the quantity of the plasmids were
reviewed with a Nanodrop (Materials #41).
Next, plasmids were sequenced to screen for any mutations. 7µL (1-4µg/µL) of each
plasmid was combined with 0.7µL of 0.04 µg/µL forward or reverse sequencing primer and sent
to TCAG sequencing facilities (Materials #36) at Sick Kids Hospital.
Sequencing Primers
pRTS-1-seq-forward-1
5‟-TGA CCT CCA TAG AAG ACC G-3‟
pRTS-1-seq-forward-2
5‟-CTA TCA GTG ATA GAG AAA AG-3‟
pRTS-1-seq-reverse
5‟- ATG GCC TCA CTG GCC ATT-3‟
3.2 Cell Culture – General Procedures
α-MEM (Materials # 49) and D-MEM (Materials #48) culture media were prepared in
100mL aliquots with 10% FBS (Materials #53) and 10% antibiotics cocktail (Materials #45)
unless specified otherwise. Prior to use, all media were pre-warmed in a 37oC water bath. Cells
were plated at a density of 1x105 cells/mL in T75 cell culture flasks (Materials #69) unless
specified otherwise and culture media was replaced every Monday, Wednesday, and Friday of
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the week. Cells were passaged upon 80% confluence and a 1:3 split ratio was used for
distribution.
Table 3.2.1 Cell Thawing Protocol
Step Description
1 Thaw 1mL cell vial in a 37oC water bath
2 Add 1mL of pre-warmed culture media and wait 2 minutes
3 Transfer cells to a 14mL round-bottom tubes (Materials #1)
4 Add 2mL of pre-warmed culture media and wait 2 minutes
5 Add 4mL of pre-warmed culture media (total of 8mL) and wait 2 minutes
6 Centrifuge for 5 minutes at 1500 rpm
7 Aspirate the supernatant
8 Resuspend the cell pellet with 8mL of culture media
9 Transfer 500µLof cell suspension into a cell counter isotonic solution (Materials #72) and determine cell
count
Table 3.2.2 Trypan Blue Cell Viability Protocol
Step Description
1 Prepare a 0.4% solution of trypan blue (Materials #58) in buffered isotonic salt solution, pH 7.2 to 7.3
2 Add 0.1 mL of trypan blue stock solution to 1 mL of cells
3 Load a hemacytometer (Materials #73) and examine immediately under a microscope at low
magnification
4 Count the number of blue staining cells and the number of total cells
5 % viable cells = [1.00 – (Number of blue cells ÷ Number of total cells)] × 100
3.3 HUCPVC Characterization
HUCPVCs (Materials #43) characterization was conducted with two different α-MEM
culture media and they were prepared with 10% fetal bovine serum (Materials # 53) from two
separate lots: Hyclone (Cat# SH30397.03, Lot# K4M25240) and Hyclone (Cat# SH30396.03,
Lot# KPJ22093). HUCPVCs from cords 0408003, 0508001, 0508002, 0508003 were used and
plated onto 6-well culture plates (Materials #66) at a cell density of 10 000 cells/well. Culture
media was replaced every Monday, Wednesday, and Friday. Cells were passaged at 80%
confluence and the dates were recorded and compared between cords.
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3.4 Hygromycin B Kill Curve of HUCPVC, RBMC, ROS cells
A kill curve determines the lowest possible concentration of antibiotic as well as the time
it takes to kill all untransfected cells. In order to separate transfected cells from the untransfected
cells, hygromycin B antibiotic (Materials #77) was used as the selection pressure. Hygromycin B
is an antibiotic that inhibits the growth of eukaryotic cells by interfering with protein synthesis.
HUCPVCs, RBMCs (Materials #46), and ROS cells (Materials #45) do not naturally carry the
hygromycin B resistance gene and fail to survive under its selection pressure. The pRTS-1
plasmid contains the hygromycin B resistance gene and it encodes the hygromycin B
phosphotransferase (HpH). Cells transfected with the pRTS-1 plasmid have a selective advantage
and thus, survives under selection pressure.
Three 6-well plates (Materials #66) were labeled as HUCPVCs (cord #0508003), RBMCs
or ROS cells and a 3 mm x 3 mm area was marked below each well. These areas were marked to
ensure consistent daily data collection. Ten thousand cells were seeded into each well of their
respective plates and cultured to 80% confluence in the absence of hygromycin B. Upon 80%
confluence, corresponding hygromycin B concentrations were added to the culture media and
replaced daily. The marked region was photographed daily and cell counts of live cells were
gathered over ten days or until all cells in the region were lost.
Table 3.4 Hygromycin B Concentration Levels for Kill Curve
Well Number HUCPVC (µg/mL) RBMC (µg/mL) ROS (µg/mL)
1 0 0 0
2 5 25 25
3 10 50 50
4 25 100 100
5 50 200 200
6 1000 1000 1000
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3.5 Transfection
Five different methods were used to transfect the pRTS-1-X constructs into three cell
types and they are listed in the chronological order followed in this thesis:
3.5.1 Amaxa Nucleofector II
Nucleofection is an electrical transfection method where the negatively charged
DNA is delivered into the cell nucleus by an electrical current.64
All nucleofections were
conducted in accordance to the protocols supplied by the nucleofection kit. Each
transfection was completed with trypsinzed 1x106 cells in Nucleofection cuvette unless
specified otherwise.
First sets of nucleofection experiments were conducted with the Cell Line
Optimization Nucleofector Kit (Materials #78). All optimizations were completed with
either 2µg of pmaxGFP (3.4kbp) control plasmid (Materials #78) or 2-5µg of pRTS-1-
Luc (18.5kbp) plasmid (Materials #30). Optimization programs were tested with both
solution L and V transfection reagents. Transfected cells were transferred into 6-well
plates containing culture media and incubated for 24 hours at 37oC and 5% CO2 to allow
cell recovery. After 24 hours of incubation, the culture media was replaced with media
containing the optimized hygromycin B concentration of 50µg/mL. At this point,
transfected cells were treated with doxycycline (Materials #79) at 500ng/mL. Cells were
observed daily using a fluorescence microscope (Materials #85) to check cell viability
and GFP expression. Finally, transfection efficiencies were visually approximated using
GFP expression 24 and 144 hours after addition of dox. Furthermore, upon confluence,
activated cells were sorted using Fluorescence Activated Cell Sorting (FACS)
(Materials #88) and only the GFP expressing cells were passaged.
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Fluorescence Activated Cell Sorting (FACS)
In this research, cells were sorted using GFP, which is expressed only in pRTS-1
transfected cells. GFP sorting uses an Argon laser at 488nm excitation which
fluorescence GFP and is captured using a 525nm band pass filter of the photomultiplier
tube (PMT).
First, the passaging cells and negative, untransfected control cells were diluted to
1x106 cells/mL in α-MEM. Then the cell debris was excluded by gating 80-90% of cell
suspension based on granularity and size. Subsequently, a 1% baseline for GFP
expression was set using the negative control and using this baseline, each transfected
cell suspension was sorted from non-GFP expressing cells.
The second set of nucleofections was completed using the Human Mesenchymal
Stem Cell Nucleofector Kit (Materials #78). This kit used a different nucleofection
buffer solution and applied four different programs (A-020, C-017, P-016, and X-001).
In addition to these differences, the following suggestions were made by an Amaxa‟s
technical support to increase transfection efficiency:
a. Increase the number of cells to be transfected from 1x106 to 3x10
6 cells.
b. Increase the amount of DNA to 10 or 20 µg per reaction.
c. Perform a recovery step post-nucleofection – Transfer transfected cells
into an eppendorf tube immediately and incubate for 10 minutes prior to
plating.
d. Activate and apply selection pressure after 6 hours instead of 24 hours.
e. Check cell viability and transfection efficiency at 6/24/48 hour mark.
Cells were observed for 72 hours using fluorescence microscopy to check cell
viability and level of GFP expression.
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3.5.2 Invitrogen Lipofectamine 2000
Lipofectamine is a chemical transfection reagent that utilizes cationic lipids to deliver
DNA into the cytosol. Lipofectamine 2000 (Materials #81) protocol for adherent mammalian
cells was followed and scaled up to a 35-mm culture dish. In this protocol, HUCPVCs were first
cultured to 80% confluence in the absence of any antibiotics. Next, the lipofectamine reagent
was combined with 2µg of pRTS-1-Luc plasmid and incubated for 45 minutes at room
temperature prior to transfection. The cultured media was then replaced with the lipofectamine-
DNA complex and incubated for 5 hours. Transfected cells were induced and placed under
selection pressure after 5 hours and observed daily.
3.5.3 Qiagen SuperFect
SuperFect is a chemical transfection reagent that generates a DNA-nanoparticle complex
which is then taken into the cell through nonspecific endocytosis. Qiagen SuperFect‟s (Materials
#82) standard transfection optimization protocol for adherent cells was conducted on HUCPVCs
and RBMCs in a 24-well culture plate (Materials #64). Four different parameters were studied
for this optimization process: (1) initial number of cells, (2) amount of DNA, (3) DNA to
SuperFect reagent ratio, and (4) the incubation period. Transfected cells were induced and placed
under selection pressure immediately after transfection.
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(1) 2x104/4x10
4/6x10
4/8x10
4 cells were transfected with 1µg of DNA at 1:5
DNA to SuperFect ratio and incubated for 4 hours.
(2) & (3) 6x104 cells were transfected using the following amount of DNA and
DNA to SuperFect ratio (Table 3.5.4) and incubated for 4 hours:
Table 3.5.3 SuperFect Optimization Experiment (DNA and DNA:SuperFect Ratio)
Amount of DNA 1:2 Ratio 1:5 Ratio 1:10 Ratio
1µg DNA - 1 µg
SF - 2 µg
DNA - 1 µg
SF - 5 µg
DNA - 1 µg
SF - 10 µg
2µg DNA - 2 µg
SF - 4 µg
DNA - 2 µg
SF - 10 µg
DNA - 2 µg
SF - 20 µg
4µg DNA - 4 µg
SF - 8 µg
DNA - 4 µg
SF - 20 µg
DNA - 4 µg
SF - 40 µg
(4) 6x104 cells were transfected using 1µg of DNA at 1:5 ratio and incubated for
1/2/4/8/16 hours.
3.5.4 Clonetech Xfect
Similar to SuperFect, Xfect (Materials #83) was the final chemical transfection
reagent that was used in this research thesis and it functions by generating a DNA-
Nanoparticle complex, which is taken up by the cell. HUCPVCs were first transfected
for 4 hours using the optimization protocol at 80% confluence with 5µg of pRTS-1-X
plasmid. A second transfection was conducted on HUCPVCs, RBMCs, and ROS cells at
<50% confluence with 5µg of pRTS-1-X plasmid. Transfected cells were induced and
placed under selection pressure immediately after transfection.
3.5.5 Bio-Rad Gene Pulser MX Cell Electroporation System
The MX Cell electroporation system utilizes an electrical current to force the
negatively charged DNA into the cell. The first optimization protocol was performed on
four different cell types: human Bone Marrow cells (Materials #44), HUCPVCs,
RBMCs, and ROS cells. The optimization protocol used a preset 96-well program with a
square wave pulse and performed both voltage gradient and duration gradient
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experiments in triplicates (Figure 3.5.6). The set parameters of V-grad (100-400V) were
1000Ω resistance, 2000µf (capacitance), with a 20ms pulse duration. The set parameters
of the D-grad (10-30ms) were 1000Ω resistance, 2000µF (capacitance), and 250 volts.
This program was applied to 100µL of 1x106cells/mL with 20 µg/mL of DNA
suspended in Opti-MEM (Materials #54).
The second optimization experiment was conducted to determine the optimal
DNA concentration for transfection. The set parameters were 250 volts, 15ms, 2000µf,
1000Ω, with a square wave at a cell density of 1.5x106cells/mL. Eight different plasmid
concentrations were tested (0.5, 1, 2, 4, 8, 10, 20, 40 µg/mL) with RBMCs.
The final transfection experiment was performed to generate RBMC- and ROS-
pRTS-1-X cell systems. Twelve repeats of both cell types with each construct were
Figure 3.5.5 Diagram of the V-grad/D-grad Program – Diagram depicting the optimization protocol
experimental parameters. Rows A & E – hBM // B & F – HUCPVC // C & G – RBMC // D & H - ROS
V-grad
(100Volts)
V-grad
(200Volts)
V-grad
(300Volts)
V-grad
(400Volts)
D-grad
(10ms)
D-grad
(15ms)
D-grad
(20ms)
D-grad
(30ms)
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completed. The set parameters were 250 volts, 15ms, 2000µF, 1000Ω, with a square
wave at a cell density of 1.5x106cells/mL. Immediately following transfection, cells
were transferred to a 96-well culture plate. Cells were activated with dox 500ng/mL
(Materials #79) and placed under hygromycin B 50µg/mL (Materials #77) selection
pressure. Transfection efficiency and cell viability was observed after 24 hours.
3.6 Transfection Efficiency Analyses
Transfection efficiencies were analyzed in two ways. The first and more commonly used
method was a visual approximation of the GFP expression under a fluorescence microscope. As
stated earlier, transfected cells were activated immediately or between 4 to 24 hours post-
transfection. Cells were observed on a regular 24 hour interval unless specified otherwise. Three
positions of each well of each transfection were marked and photographed in visible and blue uv
light (FITC – 488nm). Images were manually quantified to approximate transfection efficiency.
Second and more accurate transfection efficiency were conducted between passages using FACS
as described in section 3.5.1.
3.7 Single clone Isolation
Single clones were isolated from cells transfected with the Bio-Rad electroporator
(Materials #84). Transfected RBMCs and ROS cells were selected for 7 days and 12 clones were
isolated into a 96-well plate (Materials #62) through limited dilution. Clones were cultured to
confluence under selection and visually inspected for GFP expression. Three clones with the
brightest GFP expression were selected and cultured for all subsequent experiments.
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3.8 mRNA Expression
Clones of each cell-construct combination were visually inspected to isolate clones that
retained GFP expression over time. Viable clones were cultured to 80% confluence in T75
culture flasks. The three most active clones of each construct were selected and lysed using 4mL
of cell lysis reagent (Materials #89). mRNA was then extracted using a Sigma-Aldrich GenElute
Direct mRNA Miniprep Kit (Materials #90). The extracted mRNA was converted into cDNA
using High Capacity RNA-to-cDNA Master Mix with „No RT Control‟ (Materials #91).
Constructed RT-cDNAs and No RT-cDNAs were PCR amplified using primers designed
specifically to contain a part of our gene as well as the 3‟ UTR within the plasmid. The amplified
product is approximately 150-300bp and this specific design ensured that, if present, the
amplified product was a result of our cDNA not a contamination by genomic DNA. At the same
time as the RT and No RT control, β-actin was replicated as a positive control. The following are
the sequences of the primers designed for confirmation.
BMP2-cDNA
Forward: CAT GCC ATT GTT CAG ACG TT
Reverse: CAG GTC GAG GGA TCT CCA TA
RUNX2-cDNA
Forward: CCA GAA TGA TGG TGT TGA CG
Reverse: CAG GCG TAC GGG ATC TTC
SP7-cDNA
Forward: GGA AGA GGA GGC CAG TCA G
Reverse: AGG CGT ACG GGA TCT TCC
VEGF-A-cDNA
Forward: CAG CGG AGA AAG CAT TTG TT
Reverse: CAG GTC GAG GGA TCT CCA TA
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3.9 Western Blot Analyses
To detect the expression of our gene at a protein level, the conditioned media of BMP2
and VEGF-A were collected and whole cell lysates of SP7 and RUNX2 were collected using
4mL of cell lysis reagent (Materials #89) with 25µL of Protease Inhibitor Cocktail (Materials
#92).
The protein extracts were separated on a SDS-polyacrylamide gel using a western blot.
The proteins were separated on a 12% PAGE (BMP2, OSX, RUNX2) or 15% PAGE (VEGFA)
for 1 hour and 10 minutes at 120V, then blotted onto an Immobilon-P membrane (Materials
#109) using a semi-dry transfer apparatus (Materials #121) at 100mA per gel for 1 hour.
Once transferred, the membrane was blocked with 10% skim milk powder (Materials
#115) in TBS-T (Materials #117) at 4oC, overnight, to prevent non-specific binding. The
membrane was rinsed twice with TBS-T, and then incubated with the corresponding primary
antibody (Materials #112) for 1 hour with 25% skim milk powder in TBS-T. Unbound primary
antibody was removed from the membrane and washed six times with TBS-T for 5 minutes each
time. The membrane was then incubated with the corresponding secondary antibody (Materials
#107/108) for 1 hour with 25% skim milk powder in TBS-T. Unbound secondary antibody was
removed from the membrane and washed six times with TBS-T for 5 minutes each time. The
HRP signal was activated using an ECL western blot detection kit (Materials #96/104) and the
chemiluminescent signal was captured on an 8” x 10” X-ray film (Materials #100). The X-rays
were developed and scanned into the computer. For dose-response and kinetic studies, westerns
blots were quantified using the ImageJ software (Materials #119) and plotted in Excel.
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3.10 Doxycycline Dose-Response
To determine the dose-response, stable clones were cultured without doxycycline for
more than 18 days to stop GFP expression. These clones were then plated into 6-well plates at a
density of 1x104 cells/well and cultured to 80% confluence and activated with 0, 50, 100, 250,
500, and 1000ng/mL of doxycycline. The conditioned media (BMP-2) or the cell lysates
(RUNX2 and SP7) were collected after 24 hours. Using the methods described in section 3.9,
western blots were completed and quantified through imageJ to analyze the dose-dependent
effects of doxycycline concentration.
3.11 Induction Kinetics
For short term induction analysis, non-induced transfected cells were plated into a 6-well
plate and cultured to 80% confluence. Transcription of all constructs was activated using
500ng/mL of doxycycline. Either conditioned media (BMP-2) or cell lysates (OSX and RUNX2)
were obtained at 0, 1, 2, 4, 8, 24, 72, and 144 hour time points. Samples at each time points were
collected and preserved in the -80oC freezer until all time points were obtained. Using the
methods described in section 3.9, western blots were completed. Each western blot contains
samples from all six time points and one sample of cells that have always been exposed to
doxycycline as the positive control lane. This blot was then quantified through imageJ and the
induction kinetics was studied.
For long term induction study, a preliminary study was completed. Doxycycline was first
removed from the culture media of pRTS-1transfected RBMC cells. The GFP expression was
observed daily until the signal diminished and cells were re-exposed to doxycycline.
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3.12 Alkaline Phosphatase Assay
To verify the osteogenic effects the pRTS-1-X constructs should induce, an alkaline
phosphatase activity assay in C2C12 (Materials #133) cells. Two types of ALP assay were
completed. The first method transferred a concentrated conditioned media of the activated cells
to an 80% confluent C2C12 plate. The second experiment was to co-culture the activated cells
along with the C2C12 cells. For all C2C12 assays, the media of the transfected cells were
changed to remove hygromycin B as it would kill the C2C12 cells upon transfer.
10,000 C2C12 cells were first plated and cultured to 80% confluence. Next, 4mL of
conditioned media of all cell-construct combination was placed into an Amicon Ultra Centrifugal
Filter (30k) device (Materials #97) and centrifuged for 10 minutes at 13, 257 rcf. The
concentrated conditioned media was then transferred to the C2C12 cells with 50 µg/mL of L-
Ascorbic Acid (Materials #126). Additionally, rhBMP2 200ng/mL (Materials #132), conditioned
media from pRTS-1-Luc transfected cells, and conditioned media from untransfected cells were
tested on C2C12 cells as controls.
After 3 days of incubation period the cells were rinsed twice with PBS and fixed twice
with 10% formalin (Materials #123) and twice with 4% Paraformaldehyde (Materials #128) for
10 minutes each. Cells were then rinsed again with PBS and dH20 and incubated in ALP solution
(Materials #123/124) for 30 minutes. The colour development was stopped by rinsing the cells
with PBS and dH20.
In the co-culture experiment, 1x105 cells of each C2C12 and stably transfected cells were
plated into 6-well plates and cultured for 3 days in culture media without hygromycin B. After 3
days of incubation, the same fixation and development protocol was conducted.
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Chapter 4 – Results
This chapter reports all of the results from the pRTS-1-X plasmid transfections. First, the
construct verification is reported followed by characterization of the three cell types used in this
research. The variation between the different cords of HUCPVCs is first characterized followed
by the hygromycin B kill curve of all three cell lines. Next, the results of all five transfection
methods are presented. This includes the difference between transient and stable transfection as
well as the affected proliferation rate due to transfection. Expression of transfected genes in
stable isolated clones is then analyzed at mRNA and protein levels. Finally, results of the
induction kinetics as well as the doxycycline dose-response studies are reported and the section
concludes with an analysis of alkaline phosphatase activity in the transfected cells.
4.1 pRTS-1-X Construct Verification
pRTS-1-X constructs were generated using the methods stated in section 3.1. As seen in
the figure 4.1.1, the 3‟ overhang that exists after removal of the luciferase gene by digestion with
Sfi I consist of three bases, CAC, on both ends of the pRTS-1 plasmid and do not complement
each other. This ensures that the plasmid cannot be religated without an insert that complements
the 3‟ overhang.
>>>>>>
5‟---GGCCTCAC TGGCC------luciferase-------GGCCAGTG AGGCC---3‟
3‟---CCGGA GTGACCGG--------------------------CCGGT CACTCCGG---5‟
<<<<<<
GGCCTCAC AGGCC
GGCCA CACTCCGG
Figure 4.1.1 Sfi I Restriction Sequence in the pRTS-1-Luc Plasmid and its Unique 3’ Overhang – Sfi I
restriction sites are indicated in red; the resulting 3‟ overhang (Blue) prevents self relegation of the linearized
plasmid.
3‟
3‟
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The disadvantage of having a Sfi I restriction site is that the gene of interest can be
inserted in either direction and as a result, the correct orientation must be confirmed through
diagnostic digests and sequencing (See section 3.1.5).
g
Cloning was accomplished using the techniques mentioned in section 3.1. The pRTS-1-
RUNX2 and pRTS-1-SP7 plasmids were confirmed to have the correct size and orientation of
insert (figure 4.1.2). Plasmids were further verified for point mutations through sequencing. The
quality of sequencing data for the pRTS-1-VEGF-A plasmid returned below threshold and thus
could not be confirmed.
4.2 HUCPVC Characterization
HUCPVCs from four different cords were tested to see if there is variability between
cords. Cells exhibited “fibroblast-like” appearance with stellate shape and long cytoplasmic
processes. After culturing HUCPVCs for over 10 passages, the only notable difference found
Figure 4.1.2 Confirmation of Insert Orientation by Diagnostic Digest – (A) Uncut pRTS-1-Luc, (B) pRTS-1
digested with Sfi I, (C) Sense orientation of pRTS-1-RUNX2 digested with Not I, (D) Antisense orientation of
pRTS-1-RUNX2 digested with Not I, (E) Sense orientation of pRTS-1-RUNX2 digested with Hind III, (F)
Antisense orientation of pRTS-1-RUNX2 digested with Hind III, (G) Sense orientation of pRTS-1-OSX digested
with Bgl II, (H) Antisense orientation of pRTS-1-OSX digested with Bgl II, (I) Sense sized insert of VEGF-A
digested with Sfi I.
H G
1450bp
2600bp
1600bp
2000bp
1500bp
1000bp
500bp
3000bp 4000bp 5000bp 6000bp 8000bp
10000bp
A B
2000bp
1500bp
1000bp
500bp
3000bp 4000bp 5000bp 6000bp 8000bp
10000bp
C D
7500bp
6400bp
2000bp
1500bp
1000bp
3000bp
4000bp
5000bp
6000bp 8000bp
10000bp
F E
5540bp 5430bp
700bp
800bp
pRTS-1 RUNX2 OSX VEGF-A
200bp
300bp
400bp
500bp
650bp
850bp
1000bp
1650bp
2000bp
5000bp
12000bp
100bp
500bp
I
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56
was the number of passages the cells survived. Cells from cords 0508001 proliferated past 10
passages while cells from cord 0408003 did not proliferate past the 6th
passage.
4.3 Hygromycin B Kill Curve of HUCPVC, RBMC, and ROS cells
The lowest concentration of hygromycin B to kill all non-transfected cells was
determined as described in section 3.4. The results are as follows:
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Ap
pro
xim
ate
Pe
rce
tage
of
Via
ble
Ce
lls
HUCPVC Kill Curve (Hygromycin B)
0 µg/mL
5 µg/mL
10 µg/mL
25 µg/mL
50 µg/mL
1000 µg/mL
Figure 4.3.1 HUCPVC Kill Curve - The minimal hygromycin B concentration to kill all cells within 7
days was determined to be 50 µg/mL.
Figure 4.2 HUCPVC characterization – (A) Cord 0408003, (B) Cord 0508001, (C)
Cord 0508002, (D) Cord 0508003. Minimal variability in cell proliferation was
observed between cords.
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All HUCPVCs were killed within four days of period at 50µg/mL while RBMCs and
ROS cells were killed in seven days using 25µg/mL and 50µg/mL respectively. As a result, all
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Ap
pro
xim
ate
Pe
rce
tage
of
Via
ble
Ce
lls
RBMC Kill Curve (Hygromycin B)
0 µg/mL
25 µg/mL
50 µg/mL
100 µg/mL
200 µg/mL
1000 µg/mL
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
Ap
pro
xim
ate
Pe
rce
tage
of
Via
ble
Ce
lls
ROS Kill Curve (Hygromycin B)
0 µg/mL
25 µg/mL
50 µg/mL
100 µg/mL
200 µg/mL
1000 µg/mL
Figure 4.3.2 RBMC Kill Curve – The minimal hygromycin B concentration to kill all cells within 7
days was determined to be 25 µg/mL.
Figure 4.3.3 ROS Kill Curve - The minimal Hygromycin B concentration to kill all cells within 7 days
was determined to be 50 µg/mL.
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selection processes for transfection experiments were completed with 50µg/mL of hygromycin B
to select for cells transfected with the pRTS-1 vectors.
4.4 Transfection
Three critical factors were studied for each transfection method: cell survival post-
transfection, transfection efficiency, duration of expression after transfections (i.e., transient vs.
stable transfections).
4.4.1 Amaxa Nucleofector II
Initial transfections of HUCPVCs with the pRTS-1 plasmid using the optimization kit
showed that the program used for each transfection has an impact on cell survival. Programs A-
020, T-020, T-030, X-001, X-005, L-029, and D-023 were tested with both Nucleofection
solutions (L and V). Cell survival post-transfection with the pRTS-1 Vector is as follows (figure
4.4.1.1):
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Figure 4.4.1.1 Phase Contrast of Cells 24 Hours Post-Transfection and Fluorescence after 24
Hours of Doxycycline Induction – HUCPVCs transfected using Nucleofection with the large pRTS-
1 vector (18.5kbp) kills large number of cells in both solution L and V.
Nucleofection Solution V
Program Phase-Contrast (24 hours post transfection)
Fluorescent (24 hours post dox induction)
Phase-Contrast (24 hours post transfection)
Fluorescent (24 hours post dox induction)
Nucleofection Solution L
A-020
D-023
L-029
T-020
T-030
X-001
X-005
No Pulse
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60
In comparison to the no pulse control, HUCPVC viability was compromised by all
programs suggested in the Optimization Kit (Materials #78). Minimal discrepancy was found
between solution L and V. GFP expression was not observed in any of the transfected cells after
24 hours of dox induction (figure 4.4.1.1). Cultures of all programs with the exception of X-001
were killed after four days of selection (figure 4.4.1.2).
Program X-001 with solution V produced marginally higher cell viability than solution L.
GFP fluorescence was observed after four days of exposure to doxycycline in both solutions L
and V cultures (figure 4.4.1.3). Cells from solution V did not proliferate and died after 9 days of
selection, indicative of low, but transient transfection efficiency.
Program
Phase-Contrast (4 Days post-selection)
Nucleofection Solution L
A-020
D-023
L-029
T-020
Nucleofection Solution V
Phase-Contrast (4 Days post-selection)
Nucleofection Solution L
Nucleofection Solution V
Program
Figure 4.4.1.2 Phase Contrast of HUCPVCs after Four Days of Selection – Programs A-020, D-023, L-029, T-
020, T-030, and X-005 transfected cells did not produce viable cells after four days of selection. X-001 and the no
pulse control cells proliferated post-selection.
X-005
T-030
X-001
No Pulse
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HUCPVCs transfected in solution L with program X-001 began expansion after 20 days
of selection but GFP expression decreased over every passage (figure 4.4.1.4). The first passage
of cells was from a 10cm culture dish to a T75 flask and was excluded from FACS. In
subsequent passages, cells were sorted through FACS upon confluence to enrich the GFP
expressing population of cells and cultured in T75 flasks.
Figure 4.4.1.4 Diminishing GFP Expression of Nucleofected HUCPVCs – Cells transfected with the pRTS-
1-Luc plasmid in solution L with program X-001 proliferated after 20 days but FACS indicates a diminished
GFP expression at every passage.
Nucleofection Solution L
X-001
Nucleofection Solution V
Phase-Contrast Fluorescent Phase-Contrast Fluorescent Program
Figure 4.4.1.3 HUCPVCs transfected (Program X-001) with the pRTS-1 Plasmid Expressing
GFP – GFP is observed from transfected cells after being exposed to doxycycline for four days.
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Pe
rce
nta
ge o
f G
ate
d G
FP E
xpre
ssin
g C
ells
(%
)
Passage Number
Diminishing GFP Expression of HUCPVCs Transfected with pRTS-1 Plasmid in Solution L Using Program X-001
HUCPVC(X-001,Solution L)
Passage #1: - 25 days after transfection. - Cells passaged from 10cm culture dish into T75 flasks
Passage #2: - 31 days after transfection. - Cells passaged into new T75 flasks
36 day
40 days
44 days
49 days
55 days
59 days
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62
A second set of Nucleofection was completed with the Human Mesenchymal Stem Cell
Kit. As discussed in section 3.5.1, modifications were made to the protocol in attempts to
increase cell viability and transfection efficiency. Nucleofection results of the MSC kit (figure
4.4.1.5[A-D]) using the pRTS-1-Luc plasmid were similar to the results of the optimization kit
(figure 4.4.1.1). Cell viability post-transfection was negligible even with the increased number of
initial cells. Conversely, transfection with the control plasmid (3kbp) produced high transfection
efficiency.
Figure 4.4.1.5[A] Modified MSC Kit Nucleofection Protocol with Program A-020 – Despite a three-
fold increase in the initial number of transfected cells, cell viability and transfection efficiency of the
pRTS-1 plasmid is low.
24 Hours Post-Nucleofection
Control Plasmid
48 Hours Post-Nucleofection
Phase-Contrast Fluorescent Phase-Contrast Fluorescent Plasmid
10µg of pRTS-1 Plasmid
20µg of pRTS-1 Plasmid
[A] MSC Kit – Program A-020
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24 Hours Post-Nucleofection
Control Plasmid
48 Hours Post-Nucleofection
Phase-Contrast Fluorescent Phase-Contrast Fluorescent Plasmid
10µg of pRTS-1 Plasmid
20µg of pRTS-1 Plasmid
[B] MSC Kit – Program C-017
Control Plasmid
10µg of pRTS-1 Plasmid
20µg of pRTS-1 Plasmid
[C] MSC Kit – Program P-016
Figure 4.4.1.5[B-C] Modified MSC Kit Nucleofection Protocol with Program C-017 [B] and P-016 [C]
– Despite a three-fold increase in the initial number of transfected cells, cell viability and transfection
efficiency of the pRTS-1 plasmid is low.
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64
In figures 4.4.1.5[A-D], the cell viability after 24 hours appears to be higher in
comparison to the optimization kit (figure 4.4.1.1) but this may be the result of a three-fold
increase in the number of cells transfected. Thus the images presented in figure 4.4.1.5 [A-D] do
not give a correct visual representation of cell viability. It is also important to note the brightness
of GFP between the control plasmid and the pRTS-1-Luc plasmid. HUCPVCs transfected with
the control plasmid exhibit bright GFP fluorescence for all programs but the pRTS-1-Luc
transfected cells have minimal GFP fluorescence. Table 4.4.1 is a summary of the transfections
completed with Amaxa‟s Nucleofector.
24 Hours Post-Nucleofection
Control Plasmid
48 Hours Post-Nucleofection
Phase-Contrast Fluorescent Phase-Contrast Fluorescent Plasmid
10µg of pRTS-1 Plasmid
20µg of pRTS-1 Plasmid
[D] MSC Kit – Program X-001
Figure 4.4.1.5[D] Modified MSC Kit Nucleofection Protocol with Program X-001 – Despite a three-
fold increase in the initial number of transfected cells, cell viability and transfection efficiency of the
pRTS-1 plasmid is low.
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Table 4.4.1 Summary of Nucleofection Experiments
Date Parameters Result
March 3rd,
2009
Optimization Kit
Program: X-001
Cell Type: HUCPVC
# of Cells: 1.5x106
Plasmid Used: 2µg of pRTS-1
Nucleofection Solution: L and V
No surviving cells for solution L
Solution V shows low transfection
efficiency with massive cell death after 3
days
All cells are dead within 1 week with no
expansion
March 10th,
2009
Optimization Kit
Program: X-001
Cell Type: HUCPVC
# of Cells: 1.0x106
Plasmid Used: 5µg of pRTS-1
and 2µg of Amaxa Plasmid
Nucleofection Solution: L and V
Solution V – transient transfection (death
after 9 days)
Solution L – Proliferated but no GFP
expression seen until 4th day of induction
Transfection efficiency drops from 6% to
1% between 6 passages
Amaxa plasmid (3kb) shows high
efficiency
March 23rd,
2009
Optimization Kit
Program: A-020, T-020, T-030,
X-005, L-029, D-023
Cell Type: HUCPVC
# of Cells: 1.0x106
Plasmid Used: 3µg of pRTS-1
Nucleofection Solution: L and V
Massive cell death during transfection
No GFP expression
Surviving cells killed off during selection
period
May 28th,
2009
MSC Kit
Program: X-001
Cell Type: HUCPVC
# of Cells: 1.0x106
Plasmid Used: 5µg of pRTS-1
Low cell survivability post-transfection
No stable transfected cells found
August
17th, 2009
MSC Kit
Program: U-23, C-17, P-016, X-
001
Cell Type: HUCPVC
# of Cells: 1.0x106
Plasmid Used: 3µg of pRTS-1,
2µg of control plasmid(Amaxa)
Control plasmid has high survivability and
high efficiency through all programs
C-17, P-016 fails to have any viable cells
U-23 low cell survivability with single cells
expressing GFP after 5 days of selection
U-23 plate does not proliferate
March 29th,
2010
MSC Kit
Program: U-23, C-17, P-016, X-
001
Cell Type: HUCPVC
# of Cells: 3.0x106
Plasmid Used: 10µg, 20µg of
pRTS-1, 10µg of control
plasmid(Amaxa)
Control plasmid has high survivability and
high efficiency through all programs
Low cell survivability with all programs
using the pRTS-1 plasmid
No visible GFP expression
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66
4.4.2 Invitrogen Lipofectamine 2000
Invitrogen‟s liposomal-based transfection method failed to achieve any transfection with
HUCPVCs. Cell viability was unaffected by the transfection but the cells were killed during
antibiotic selection. Furthermore, no visible GFP expression was observed after 24 hours of
exposure to doxycycline (figure 4.4.2). These results indicate that no transfection took place.
4.4.3 Qiagen Superfect
As described in section 3.5.3, four parameters were optimized: initial number of cells,
amount of DNA, DNA to SuperFect reagent ratio, and the transfection period. The cell density at
the time of transfection did not affect the results. Increasing the amount of DNA and the
Superfect-to-DNA ratio as well as the incubation period, all increased the transfection efficiency
without having an adverse effect on cell survivability. Superfect produced the highest
transfection efficiency with RBMCs of all methods used in this research and GFP expression was
observed as early as 12 hours post-activation (figure 4.4.3.2) while HUCPVCs showed minimal
transfection efficiency (figure 4.4.3.1). Under selection, HUCPVCs did not survive past 4 days.
RBMCs survived for seven days, which is approximately three days longer than selection, thus,
indicative of a transient transfection.
Figure 4.4.2 Lipofectamine 2000 Transfection of HUCPVCs – (A) HUCPVC is killed over four
days of selection pressure. (B) GFP fluorescence is absent post-transfection. The lack of GFP and
the rate of cell death suggest that no transfection was achieved.
Day 0
Day 0
(A)P
hase
-Contr
ast
(B)F
luore
scent
Day 1
Day 1
Day 2
Day 2
Day 3
Day 3
Day 4
Day 4
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67
HUCPVC
RBMC
4.4.4 Clonetech Xfect
Initial transfections with the pRTS-1 plasmids were conducted at 80% confluence and
cells were placed under selection after four hours. GFP expression was observed in the first 12
hours of activation in RBMCs and ROS cells but not in HUCPVCs. Proliferation of ROS cells
were unaffected by Xfect while RBMCs resumed proliferation after two weeks and HUCPVCs
after three weeks post-transfection (figure 4.4.4.1). No GFP expression was found from Xfect
transfected HUCPVCs.
Figure 4.4.3.2 Qiagen SuperFect Transfection of RBMCs with Selection Pressure – (A) RBMCs are killed over seven days of selection pressure. (B) GFP fluorescence is visible as early as 12 hours post-activation but the overall number of cells decreases due to selection pressure. This data suggests a transient-only transfection was achieved.
12 Hours
12 Hours
Day 1
Day 1
Day 3
Day 3
Day 5
Day 5
Day 7
Day 7
(A)P
hase
-Contr
ast
(B)F
luore
scent
12 Hours
12 Hours
Day 1
Day 1
Day 2
Day 2
Day 3
Day 3
Day 4
Day 4
(A)P
hase-C
ontr
ast
(B)F
luore
scent
Figure 4.4.3.1 Qiagen SuperFect Transfection of HUCPVCs with Selection Pressure – (A) HUCPVCs are killed over four days of selection pressure. (B) GFP fluorescence is absent post-transfection. The lack of GFP and the rate of cell death suggest that no transfection was achieved.
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68
g
Transfections Completed at 80% Confluent Culture Dish
(24 Hours Post-Transfection)
HUCVPC
Transfections Completed at <50% Confluent Culture Dish
(Three Weeks Post-Selection)
Phase-Contrast Fluorescent Phase-Contrast Fluorescent
Cell
RBMC
ROS
Figure 4.4.4.1 Xfect Transfected Cells Post-Selection – HUCPVCs do not proliferate but survive
past selection without GFP expression. RBMC and ROS cells express GFP post-selection and
continue to proliferate.
Figure 4.4.4.2 Long-Term Decrease in GFP Expression of Xfect Transfected RBMC and ROS –
Once the selection pressure is removed, both RBMCs and ROS cells lose GFP Expression over time.
One week Post-Transfection Five Months Post-Transfection
Phase-Contrast Fluorescent Phase-Contrast Fluorescent
Cell
RBMC
ROS
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69
Cell viability was unaffected by the reagent but the level of confluence at the time of
transfection did affect the overall viability. Cells transfected at 80% confluence formed large
complexes that look like cell disintegration (figure 4.4.4.3). Under higher magnification, visible
changes to the cell morphology were observed. Transfected cells expressed GFP but massive cell
death was observed and cells began to peel off the culture dish. A secondary repeat of the
experiment confirmed this phenomenon for all cell types.
Repetition of the Xfect transfection using 50% confluence presented a different long-term
result for RBMC and ROS cells. The viability was unaffected by the chemical reagent and the
formation of large complexes and cellular disintegration was not observed (figure 4.4.4.2).
Transfection efficiency was approximately 10% after 24 hours of activation (figure 4.4.4.4).
Proliferation of ROS cells were unaffected by transfection but RBMCs began proliferation after
2 weeks of delay under selection. GFP expressing cells beyond the selection period were isolated
into single clones. These clones proliferated for a 5 month period under constant hygromycin B
Figure 4.4.4.3 Formation of Large Complexes and Cell Disintegration Caused by High
Cell Density during Transfection – Xfect transfection at high (80%) cell confluence cause
major changes to the cell.
(A)P
hase
-Contr
ast
(B)F
luore
scent
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70
selection but once the selection was removed, the cells‟ GFP expression level diminished (figure
4.4.4.2).
4.4.5 Bio-Rad Gene Pulser MX Cell Electroporation System
The first optimization process using the preset 96-well program provided a wide range of
results between the different cell types. HUCPVC and human Bone Marrow (hBM) cells had no
cell attachment after transfection through all voltage and duration gradients while RBMC and
ROS cells produced viable cells. Transfected RBMC and ROS cells expressed GFP after 12
hours (figure 4.4.5.1) and proliferated within one week of transfection. From these cells the
following optimization data were obtained:
Table 4.4.5.1 – Bio-Rad Electroporation Voltage Gradient Results
Voltage (V) Results
100 Cells survived transfection, but, no live cells found after 8 days of selection
200 GFP expression found for ROS and RBMCs and passaged after 8 days of selection
300 GFP expression found for ROS and RBMCs and passaged after 8 days of selection
400 All cells were killed during transfection
Table 4.4.5.2 – Bio-Rad Electroporation Pulse Length Gradient Results
Duration (ms) Results
10 Low cell survivability (<1%)
15 High cell survivability (>50%)
20 High cell survivability (>50%)
30 High cell survivability (>50%)
(A)Phase-Contrast (B)Fluorescent
Figure 4.4.4.4 RBMCs Transfected with Xfect at 50% Confluence
Results in Low Transfection Efficiency – Transfection efficiency after 24
hours of dox induction is approximately 10%.
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71
The result of the second transfection experiment indicated that 4 µg/mL of pRTS-1
plasmid is optimal for electroporation and no significant improvement was observed beyond this
concentration (figure 4.4.5.2).
Repetition of these experiments confirmed the optimum transfection condition to be a
square wave pulse at 250V for 15ms with 4 µg/mL of DNA in 1.5x106cells/mL. Using this
optimized condition, all pRTS-1-X constructs were transfected in both RBMC and ROS cells.
12 Hours Post-Transfection
Phase-Contrast Fluorescent
Cell
RBMC
ROS
Figure 4.4.5.1 Bio-Rad Transfected RBMCs and ROS Cells Express GFP after 12 Hours
0.5 µg/mL 1 µg/mL [DNA]
RBMC
2 µg/mL 4 µg/mL
8 µg/mL 10 µg/mL 20 µg/mL 40 µg/mL
Figure 4.4.5.2 Bio-Rad DNA Concentration Optimization Transfection – Transfection efficiency is
increased by increasing the transfecting DNA concentration to 4 µg/mL. Further increase appears to reduce
transfection efficiency.
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These cells were isolated into stable clones and used for all analyses found in the following
sections.
4.5 Transient vs. Stable Expression and Production of Transfected Clones
All transfection methods mentioned in the previous sections achieved a certain degree of
transfection with the exception of lipofectamine 2000. In the electrical transfection method,
Amaxa‟s Nucleofector produced transient-only transfected HUCPVCs that did not proliferate
while Bio-Rad‟s electroporator produced stably transfected RBMC and ROS cells for all pRTS-
1-X constructs. From these stably transfected cells, individual clones were isolated through
limited dilution (figure 4.5.1 and figure 4.5.2 C&D) and they retained the GFP expression for 8
months in the absence of a selection pressure, thus, indicative of a stable transfection.
The two chemical transfection methods (excluding Lipofectamine) achieved higher
transient transfection efficiencies than electroporation methods. Xfect transfected RBMC (figure
4.5.2 A&B) and ROS cells proliferated and single clones were isolated. These clones were
believed to be stable but when the selection pressure was removed after 5 months, cells rapidly
lost GFP expression in the presence of doxycycline. Qiagen Superfect produced the highest
Figure 4.5.1 Single Clone Expansion of ROS-pRTS-1-BMP-2 After 13
Days – Image of a single ROS-pRTS-1-BMP-2 clone after 13 days of
expansion
(A)Phase-Contrast (B)Fluorescent
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transient transfection efficiency with RBMCs but was unable to proliferate. As a result, stable
single clone were isolated only from RBMC and ROS cells transfected by the Bio-Rad
electroporator.
4.6 Rate of Proliferation
The rate of proliferation of each cell type was affected by the plasmid and the method of
transfection. Transfections were performed with the pRTS-1 plasmid (~18.5kbp) and the GFP
control plasmid (3kbp). Following transfections, dates of cell passages were compared. The
control plasmid did not affect proliferation in any cell type for all transfection methods while
HUCPVCs transfected with pRTS-1 using Nucleofection (program X-001 in solution L) began
proliferation after 20 days of culture. HUCPVCs transfected with the pRTS-1 plasmid using
Xfect proliferated after 21 days while RBMCs proliferated after 14 days. RBMCs and ROS cells
transfected with electroporation showed no delay in proliferation post-transfection.
Figure 4.5.2 Xfect Transfected and Electroporated RBMC - Single Clone
Expansion – Xfect transfected RBMCs single clone isolation (A&B). Single clone
expansion of Bio-Rad transfected RBMCs (C&D).
A B
C D
(A)Phase-Contrast (B)Fluorescent
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4.7 mRNA Expression
It is important to note that all analyses from this point were conducted with RBMC and
ROS cells only as expansion of transfected HUCPVCs was not possible.
The PCR amplified 150-300bp fragments generated from cDNA of each clone are shown
in figure 4.7. At the mRNA level, transfected genes are expressed in both RBMC and ROS cells
upon induction. The quantity of expression may vary between individual clones but the specific
primer design ensures that the PCR products are from the pRTS-1-X plasmid and not from an
endogenous gene expression (figure 4.7).
4.8 Western Blot Analyses
Correct protein expression of BMP2 (45kDa), RUNX2 (57kDa), and SP7 (46kDa) was
obtained from dox exposed RBMC and ROS clones. Furthermore, clones not exposed to Dox
(after 18+ days) did not express the genes of interest at a detectable level (figure 4.8). Transcripts
of pRTS-1-VEGF-A transfected constructs were detected at an mRNA level but failed to be
detected at a protein level for both cell types.
Figure 4.7 mRNA Expression of All Genes from RBMC and ROS Clones – The expression level of
each gene of interest is low in comparison to the β-actin control. Visible bands in the no RT controls
indicate genomic contamination.
RT
-BM
P2
No R
T-B
MP
2
RT
-β-a
ctin
No R
T-β
-actin
RT
-RU
NX
2
No R
T-R
UN
X2
RT
-β-a
ctin
No R
T-β
-actin
RT
-β-a
ctin
No R
T-β
-actin
RT
-β-a
ctin
No R
T-β
-actin
RT
-SP
7
No R
T-S
P7
RT
-VE
GF
-A
No R
T-V
EG
F-A
ROS
RBMC
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4.9 Doxycycline Dose-Response
Six different doxycycline concentrations were used to induce to RBMC-pRTS-1-X and
ROS-pRTS-1-X cells that have been cultured in the absence of doxycycline for more than 18
days. Cells were exposed to dox for 24 hours and the conditioned media or the cell lysates were
collected for western blot analyses to measure the dose-response (figure 4.9).
130
95
72
55
43
34
26
72 95
55
43
34
26
17
130 170
Figure 4.8 RBMC-pRTS-1-X and ROS-pRTS-1-X Western Blot
(cell lysates) -
(“+”, “-” indicates presence or absence of dox 500ng/mL)
A – ROS-pRTS-1-BMP2 (Size: 45 kDa)
B – RBMC-pRTS-1-BMP2 (Size: 45 kDa)
C – ROS- pRTS-1-RUNX2 (Size: 57 kDa)
D – RBMC-pRTS-1-RUNX2 (Size: 57 kDa)
E – ROS-pRTS-1-SP7 (Size: 46 kDa)
F – RBMC-pRTS-1-SP7 (Size: 46 kDa)
A C 130
95
72
55
43
34
26
B
72 95
55
43
34
26
17
130 170
170 130
95 72 55 43
34
26
D
E F
+
+ +
+ + +
ROS-BMP2 RBMC-BMP2 ROS-RUNX2 RBMC-RUNX2
ROS-SP7 RBMC-SP7
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0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6No
rmal
ize
d p
rote
in E
xpre
ssio
n (
%)
Doxycycline Concentration
ROS Cells Doxycycline Dose-Response (24 Hours)
BMP-2
RUNX2
SP7
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6No
rmal
ize
d p
rote
in E
xpre
ssio
n (
%)
Doxycycline Concentration
RBMC Doxycycline Dose-Response (24 Hours)
BMP-2
RUNX2
SP7
Figure 4.9 RBMC and ROS Cells Doxycycline Dose-Response after 24 Hours – A difference in GFP
expression is observed as the doxycycline concentration is increased. Transfected cells reach an upper
plateau of GFP expression at 24 hours with 500ng/mL of doxycycline.
Cell-Construct
0 n
g/m
L
50 n
g/m
L
100 n
g/m
L
250 n
g/m
L
500 n
g/m
L
1000
ng/m
L
Contro
l*
RBMC-pRTS-1-BMP2
RBMC-pRTS-1-SP7
ROS-pRTS-1-BMP2
ROS-pRTS-1-RUNX2
ROS-pRTS-1-SP7
RBMC-pRTS-1-RUNX2
* Cell lysates obtained
from the same clones
of each respective
constructs. Control
cells have been
continuously exposed
to 500ng/mL of dox
since transfection.
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4.10 Induction Kinetics
The induction kinetics of the pRTS-1-X transfected RBMC and ROS cells were studied
using the conditioned media and the cell lysates at each of the six time points. Visible GFP
expression is achieved 8 hours post-induction with 500ng/mL Dox and a continuous increase in
GFP expression is observed up to 72 hours (figure 4.10.1).
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0
20
40
60
80
100
0 Hour 1 Hour 2 Hours 4 Hours 8 Hours 24Hours
72Hours
144Hours
Pro
tein
Exp
ress
ion
re
lati
ve t
o 1
44
ho
urs
(%
)
Number of Hours Post Doxycycline Exposure
RBMC Induction Kinetics (500ng/mL)
BMP2
RUNX2
SP7
Poly. (Series4)
0
20
40
60
80
100
0 Hour 1 Hour 2 Hours 4 Hours 8 Hours 24Hours
72Hours
144Hours
Pro
tein
Exp
ress
ion
re
lati
ve t
o 1
44
ho
urs
(%
)
Number of Hours Post Doxycycline Exposure
ROS Induction Kinetics (500ng/mL)
BMP2
RUNX2
SP7
Poly. (Series4)
Figure 4.10.1 pRTS-1-x Transfected RBMC and ROS Cells Induction Kinetics (500ng/mL dox)
0 h
our
1 h
our
2 h
ours
4 h
ours
24 h
ours
72 h
ours
14
4 h
ou
rs
RBMC-pRTS-1-BMP2
RBMC-pRTS-1-RUNX2
ROS-pRTS-1-BMP2
ROS-pRTS-1-RUNX2
Cell-Construct
ROS-pRTS-1-SP7
8 h
ours
RBMC-pRTS-1-SP7
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A preliminary study indicated that the GFP expression of pRTS-1-X transfected RBMCs
reached a basal level approximately 18 days (figure 4.10.2 [C]) after doxycycline was removed
from the culture media and 16 days for ROS cells. Once basal level was reached, cells were
exposed to 500ng/mL of doxycycline and stable clones began expressing GFP again within 24
hours (figure 4.10.2 [D]), indicating that GFP expression could be turned “off” and repeatedly
turned “on”. This was completed three times during the course of this thesis.
4.11 Alkaline Phosphatase Assay
Alkaline phosphatase assay was completed to check for increased osteogenic activity.
When the conditioned media (CM) was transferred from the culture flasks of pRTS-1-X
transfected ROS or RBMC cells to a C2C12 plate and incubated for three days, minimal ALP
activity was observed (figure 4.11.1). BMP2, RUNX2, and SP7 conditioned media generated
three or four small patches of cells where ALP activity was shown (figure 4.11.1[A-C]). The
controls – CM of pRTS-1-Luciferase and CM of non-transfected RBMC cells did not induce
ALP activity while the rhBMP-2 as positive control produced a clear ALP activity (figure 4.11.1
[D-F]).
Figure 4.10.2 pRTS-1 Transfected RBMC Doxycycline Kinetics – RBMC-pRTS-1 clone was exposed
to 500ng/mL to doxycycline to induce GFP expression within 12 hours [A&D]. Doxycycline was
removed from the culture media and GFP expression was lost in 18 days [C].
(A)P
hase
-Contr
ast
(B)F
luore
scent
12 Hours Post-Transfection
Dox Induced Stable Clone
Dox Removed Stable Clone
Dox Re-induced Stable Clone
B C D A
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To test the autocrine and paracrine effects of transfected cells, C2C12 cells were co-
cultured with transfected RBMCs and ROS cells. In the co-culture ALP assays, RBMC-RUNX2,
RBMC-SP7, and RBMC-pRTS-1-Luc did not produce any visible ALP activity but RBMC-
pRTS-1-BMP2 co-cultured with C2C12 cells produced high ALP activity over the controls. The
ROS co-culture experiments presented strong ALP activity over all plates with no significant
distinction between the different pRTS-1-X constructs (figure 4.11.2).
Figure 4.11.1 Alkaline Phosphatase Assay (Conditioned Media):
(A) C2C12 cells exposed to RBMC-pRTS-1-BMP2 conditioned media
(B) C2C12 cells exposed to RBMC-pRTS-1-RUNX2 conditioned media
(C) C2C12 cells exposed to RBMC-pRTS-1-SP7 conditioned media
(D) C2C12 cells exposed to RBMC-pRTS-1-Luc conditioned media
(E) C2C12 cells exposed to untransfected RBMC conditioned media
(F) C2C12 cells exposed to rhBMP-2
A
D
B
E
C
F
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Figure 4.11.2 Alkaline Phosphatase Assay (Co-Culture) – All ROS co-cultures produced strong ALP activity
with no difference from the controls. RBMC-pRTS-1-BMP2 co-culture produced high ALP activity in comparison
to all other constructs and controls.
RBMC ROS
Untransfected Cells
Co-Culture
pRTS-1-Luc
pRTS-1-BMP2
pRTS-1-RUNX2
pRTS-1-SP7
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Chapter 5 – Discussion
Bone regeneration induced through direct delivery of rhBMP-2 remains problematic
because of the wasteful burst release from the carrier that creates the need for super-
physiological doses to treat non-union bone defects. Furthermore, as a result of such high doses,
there is an increased risk of complications and increased costs associated with this treatment.
Thus, it is clear that there is a need to improve the current inefficient use of expensive rhBMP-2.
By genetically engineering cells and engrafting them, a continuous expression of
osteogenic factors can be sustained.118
Furthermore, by implementing a tightly regulated control
mechanism to gene therapy, this thesis aimed to control the quantity and duration of osteogenic
factors present during bone regeneration and thus reduce the need for super-physiological doses.
Current literature has shown engraftment of genetically engineered mesenchymal stem
cell through viral transfections to be successful, although with certain limitations. Twenty six
research articles reviewed by Van Damme et al. presented transient (days) and long term (weeks
to months) expression of a transgene through viral transfections. There are five main groups of
viral vectors used for engineering: adenovirus, lentivirus, retrovirus, adeno-associated viruses
(AAV), and herpes simplex virus-1 (HSV-1).111,118
However, despite their potential for
successful gene therapy treatment, there are serious concerns related to immune response, lack of
viral vector specificity leading to dissemination of the vector, and insertional mutagenesis.111
Furthermore, viral vector preparation is a time-consuming process that requires high level of
safety.31
These issues make viral vector transfections an unsafe method of gene transfer for
engineering cells.
Alternatively, several groups attempted to address the super-physiological dose
requirement by engineering cells with the TET-On25
or TET-Off 59,82
regulation system to control
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the expression of BMP-2 in vivo. Although all three papers have shown bone regeneration, two
TET-induced/repressed methods of transfection were completed through adenoviruses and
adeno-associated two vector viruses and suffer the same issues described previously.25,82
Moutsatsos et al. used non-viral multi-vector TET-Off system and transfected C3H10T1/2 MSC
cells using Lipofectamine to control the expression of BMP-2 in vitro and in vivo.79
The latter
demonstrated controlled expression of BMP-2 with MSC cells and showed bone regeneration but
this system contains two issues that can be improved: multi-vector transfection and high
background activity.
In this thesis, a new vector was utilized to control gene expression. The pRTS-1 vector is
a one-vector system that contains both the response and reporter elements of the TET-On
system.4 Furthermore, the inclusion of a TET-repressor lowers the background gene expression
and thus allows for a tighter regulation.
During the course of the current research, five different transfection methods, both
chemical and electrical, were tested on three different cell types. While some degree of
transfection efficiency was achieved by combining specific parameters with RBMCs and ROS
cells, the project has also exposed a number of problems. Detailed explanation and the beneficial
implications of this new system will be discussed below in the context of the experimental
results.
5.1 Development of Stable pRTS-1-BMP-2 Cell System is Only Achieved with
Electroporation Combined with RBMCs and ROS cells
Three cell types were selected for incorporating the pRTS-1-BMP-2 inducible system:
two primary cell types, HUCPVC and RBMC, and an immortalized cell line, ROS. The inducible
system was incorporated into cells using chemical and electrical transfection methods and the
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results from these experiments present significant variations. These variations are dependent on
the cell type and the transfection method and they affect cell viability, cell properties, and
transfection efficiencies. Following is a summary (Table 5.1) of the transfection experiments and
their respective results:
Table 5.1 Summary of All Transfection Experiments in Chronological Order HUCPVC RBMC ROS
Amaxa
Nucleofector
II
Low viability Unexpected low viability and proliferation
problem Low transfection
efficiency
Proliferation delayed
(>2 week delay
before proliferation
resumes) or stopped
Loss of GFP
expression over
prolonged expansion
Invitrogen
Lipofectamine
2000
High viability Chemical transfection methods tested to improve
transfection efficiency No transfection
achieved
No viable cells after
selection
Qiagen
Superfect
High viability RBMC introduced to see if HUCVPC is the cause of low transfection efficiency
Low transfection
efficiency
High transfection
efficiency
Transient transfection (7 to 10 days)
No viable cells after selection
Clonetech
Xfect
High viability
Low transfection efficiency
Proliferation delayed
(>3 week delay
before proliferation
resumes)
Proliferation delayed
(>2 week delay
before proliferation
resumes)
Tested immortalized
cell line to see if
proliferation problems
could be overcome
Proliferation
unaffected by
transfection
Long-term transient transfection (~5months)
Bio-Rad Gene
Pulser MXcell
Electroporator
No viable cells
through all
parameters post-
selection
Low viability
Low transfection efficiency
Rate of expansion unaffected
Stable clones generated
Loss of expression over prolonged expansion
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These results suggest that with the current available transfection methods, it is difficult to
implement the pRTS-1-BMP-2 inducible plasmid into cells and we suggest that these variables
are mainly associated with the large size of the plasmid (18.5+kbp). The following sections
discuss in detail the chemical and electrical transfection methods and the variable results found
in the experiments.
5.1.1 Chemical Transfection Methods
Reported in this thesis are three chemical transfection methods from Clonetech,
Invitrogen, and Qiagen. Protocols achieve transfection using different mechanisms and they
produce high cell viability but low transfection efficiency. This thesis has reported that chemical
transfections achieve a transient-only state with major negative changes to the cells.
The first significant observation was the low transfection efficiency. In comparison to a
near 100% transfection efficiency with the small 3kbp control GFP plasmid (pmaxGFP plasmid,
Amaxa Nucleofection Optimization Kit), the transfection efficiencies with the pRTS-1 based
plasmids were 0.01% for HUCPVCs and between 10-50% for RBMCs, and ROS cells. Initially,
no cellular disruptions were observed but the overall number of transfected cells decreased after
a hygromycin b selection pressure was applied. These data suggests an improper transfection or a
transient-only transfection with a period shorter than that of the selection period.
As a result, to enrich the number of transfected cells, fluorescence-activated cell sorting
(FACS) was conducted at every passage level to isolate and enrich the number of transfected
cells. HUCPVCs transfected with Lipofectamine 2000 or Superfect ceased to proliferate; thus,
only Xfect transfections were isolated using FACS. Despite continuous attempts to isolate and
expand the transfected cells, results from FACS show a decrease in GFP expression over each
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passage level. These data suggest a loss of pRTS-1 plasmid from the cells and again confirm the
transient nature of chemical transfections.
The second significant observation was the diminished rate of proliferation of cells post-
transfection. Results indicated a delay or loss of proliferation for HUCPVCs while RBMCs
exhibited recovery after 14 days. This diminished rate was observed in all experimental repeats
and clones of RBMC and ROS. Furthermore, clones with resumed proliferation exhibited a
gradual loss of GFP expression over time.
Similar results were found in the literature. Hunt et al. transfected Human Umbilical Vein
Endothelial Cells (HUVEC), which are similar to HUCPVCs, with a 4.7kbp GFP-encoded
plasmid using nine different chemical transfection methods.44
Their results were similar to
HUCPVCs where the cell viability and the rate of proliferation diminished post transfection. The
authors suggested that the toxicity of the chemical reagents may alter the strength of the cell
membrane by generating reactive oxygen species, thus resulting in mass cell death.44
Upon
review, all three chemical transfections used in this thesis research contain proprietary chemical
reagents which alter cell properties for the purpose of gene insertion and an example of a change
to the cell membrane can be visualized from Xfect transfections. As seen in figure 4.4.4.3,
adjacent cells located close to one another forms large complexes. At higher magnification, these
cells appear to be disintegrated. This effect is observed when Xfect transfection is completed on
high density population of cells. The changes observed here and elsewhere44
suggest reagent
toxicity as a possible cause for low cell viability and diminished rate of proliferation.
With the chemical transfection methods currently available, stable transfections of
primary cells seem unlikely. Transient transfections may be achieved but the extremely low cell
survival makes this an inefficient approach. Furthermore, due to the transient nature of
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transfected primary cells, even if sufficient transfection efficiency is observed at 24hours, the
signal rapidly decays when assayed at 48 hours. This was observed in HUVEC transfections44
and also in HUCPVC, RBMC, and ROS cells in this research. At the present time, regardless of
the cell type, chemically induced transfections are able to achieve only transient transfections
with the large pRTS-1 plasmid.
5.1.2 Electrical Transfection Methods
Two electrical transfection methods were used: Nucleofection and Electroporation. The
results of the methods showed variations. Cell type, plasmid concentration, and transfection
conditions were the key factors that impacted the overall cell viability and efficiency of
transfection.
5.1.2.1 Nucleofection
HUCPVCs, RBMC, and ROS were transfected with the large pRTS-1 plasmid (19+kbp)
and the small control plasmid (pmaxGFP) using eleven different programs of Amaxa‟s
Nucleofector II. Two specific transfection kits were used: Cell Line Optimization Nucleofector
Kit and the Human Mesenchymal Stem Cell Kit. Both of these protocols utilize a unique buffer
to aid in transfection but showed no significant difference in results.
In this study, all three cell types showed diminished proliferation rate post transfection
with the pRTS-1 plasmid but showed no significant difference with the control plasmid. After
four days of selection and induction process, only a small percentage of HUCPVCs survived the
hygromycin B selection process and expressed GFP (<0.01%), thus making nucleofection a non-
viable method of transfection.
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Surviving HUCPVCs resumed proliferation after three weeks of inactivity and matched
the proliferation rate of a non-transfected HUCPVC. At passage number two, 6% of the cells
expressed GFP and were sorted using FACS to enrich the quantity of transfected cells.
However, by passage number eight, the number of GFP expressing cells decreased to 1.3% and
despite the continuous cell enrichment process, the expression level diminished rapidly after
each passage. These data suggest that nucleofection is unable to transfect our large plasmid to
produce stable cell clones.
As seen in Table 5.1, results of nucleofection show low viability and low transfection
efficiency. The results exhibited significant variation on cell viability in regards to the size of the
plasmid but a large difference was observed between programs. Transfection efficiencies of the
surviving cells were less than 1% with the pRTS-1 plasmid but near 100% with the control
plasmid. Furthermore, transfected cells exhibited a similar diminished proliferation rate as
observed with chemical transfection and could be explained again with Hunt et al.‟s suggestion
of chemical toxicity.44
As stated at the start of this section, two kits were used to transfect the
cells and they both contain proprietary chemical reagents which are known to be toxic to cells
upon prolonged exposure. These buffers are known to have detrimental effect on the cells and
transfection is suggested to be completed within 15 minutes of suspension in this solution.
Alternatively, because electroporation forces plasmids into cells via an electrical charge,
the strength of the charge or the size of the plasmid may damage the cell membrane and cause
the low initial viability. Upon review of the literature, the size of plasmid affects transfection
efficiencies and it has been suggested that a smaller DNA plasmid will improve cell
transfection.134
Despite nucleofection‟s enhancement of gene integration for long term
transfections, our results indicate that a stable transfection of pRTS-1 is unlikely and only a
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transient transfection is possible and only under a strict set of conditions such as those used in
this research.
5.1.2.2 Electroporation
Electroporation is an alternate transfection method with an open-ended optimization
process. An electroporator differs by allowing the user to alter the type of pulse, duration,
voltage, and resistance of each transfection. Optimization of these parameters affected the cell
viability and transfection efficiencies of pRTS-1 transfections. We found that too low voltage,
duration, or plasmid concentration all resulted in high viability but low transfection efficiency
whereas parameters that were too high resulted in massive cell death.
The results of the pRTS-1 transfections varied depending on the plasmid as well as the
cell type. HUCPVCs transfection results were identical to nucleofection. Cell viability was less
than 0.01% with diminished proliferation post-transfection. For RBMC and ROS cells, the
viability was greater than 50% and the rate of proliferation was unaffected. These data suggested
a major difference between nucleofection and electroporation.
When the protocols are compared, the main difference is the reagent the cells are
suspended in prior to and during transfection. Electroporation is carried out in a relatively inert
OPTI-MEM whereas Nucleofection is conducted in a proprietary buffer created by Amaxa. This
implies that the buffer may have a toxic effect on the cell and if so, Hunt et al.‟s suggestion of
reagent toxicity44
and its effect on cell proliferation may be true.
The second variance of electroporation was the transfection efficiency. Twenty-four
hours post-transfection and exposure to dox, GFP was expressed in RBMC and ROS cells. After
72 hours of exposure to dox and selection, GFP expression was seen in the majority of the
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surviving cells. This high expression level could be explained by the unaffected proliferation rate
and hygromycin B selection process. The selection process destroyed non-transfected cells while
the transfected cells proliferated with the resistance gene. Together, these two processes greatly
increased the number of transfected cells in 72 hours. Furthermore, the high level of GFP
expression reduced the need for FACS. Transfected cells sustained their GFP expression for 7
months before significant decrease in expression level was observed for all clones.
5.2 pRTS-1-BMP-2 Presents Low Background Expression and Doxycycline Dose-
Dependent Activation
The protein expression pattern observed in this research agreed with the data published
by Bornkamm et al.4 In the absence of dox, there is minimal background activity observed as a
result of the transcriptional silencer (tTS) and there is a doxycycline dose-dependent expression
level as observed in figure 4.9. These two observations highlight the hallmark benefits of the
pRTS-1 system, low background activity and control. In figure 4.9, the BMP-2 expression at 0
ng/mL of dox is minimal but as the concentration increases towards 1 µg/mL, an increase is
observed in all clones. This curve continues until the upper plateau is reached, where a maximum
expression level is achieved. These data coincides with those found previously.4, 30
5.3 pRTS-1 Controlled Kinetics
As discussed earlier in this chapter and by Bornkamm et al., clones exhibited variations
in the level and duration of inducibility. Clones of ROS-pRTS-1 and RBMC-pRTS-1 expressed
maximum GFP within 72 hours of exposure to doxycycline and lost its GFP expression upon
removal of doxycycline after 16-18 days. The relatively long deactivation period of GFP is due
to its long half-life and does not correspond to the expression kinetics of BMP-2.4 BMP-2 has a
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much shorter half-life in vivo.54
Thus, even if the two genes are expressed at an equivalent level
through the bidirectional promoter36
, the effects of the BMP-2 gene will not last the same
duration as the GFP.
A preliminary study on re-inducibility was tested. The sustained inducibility over time
varied between different cells and clones4 but in the absence of dox, the gene of interest was not
expressed. These non-induced clones were then re-exposed to dox and again, cells achieved
expression in 72 hours and were suppressed in 16-18 days.
Subsequently, clones were continuously cultured in an activation-deactivation cycle for
eight months. These clones exhibited continuous but diminished activity near the end of this
period where the GFP expressing number of cells diminished rapidly. Current literature cannot
explain this limited stability; thus, additional work is required to better understand this
phenomenon.
5.4 pRTS-1 Bioactivity
Bioactivity of the pRTS-1 systems were verified through an alkaline phosphatase (ALP)
activity assay. ALP assay was used to study the characteristic changes to osteogenic protein
expression in C2C12 cells as a result of the inducible system.
As expected, the positive controls showed increased ALP activity. Activated RBMC-
pRTS-1-BMP-2 generated a significant increase in ALP activity over the negative control. The
ALP activity of both negative controls (RBMC and RBMC-pRTS-1-Luc) were minimal,
suggesting that dox-exposed RBMC-pRTS-1-BMP-2 could induce osteogenesis.
In contrast, high levels of ALP activity were observed from all dox-exposed and, non-
exposed ROS-pRTS-1-X cells, as well as the negative control. Kartsogiannis et al. states that
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ROS generates a large amount of ALP activity due to its osteogenic nature57
and as a result, may
mask the effects of the dox-induced ROS-pRTS-1.
5.5 Experimental Limitations
Throughout this research thesis, our goal was to enhance gene therapy by developing a
tightly regulated, doxycycline dose-dependent cellular expression system that addressed two
major aspects of the tissue engineering triad. This system utilizes the strength of rhBMP-2in
conjunction with a novel source of stem cells, HUCPVCs, to control the expression at will. The
concept was groundbreaking and the potential for this system could be extended beyond
osteogenic gene therapy but several difficulties were faced through the course of this research
and as a result, the project turned into a proof of concept rather than a viable application for
human gene therapy.
The first major experimental limitation was the transfection. The pRTS-1 plasmid is a
large 18.5kbp plasmid which contains numerous benefits over the two-vector based Tet-On
system. However, the current methods of transfection have not been optimized to transfect large
plasmids and as a result, proved to be a major hurdle in this research. In the case of electrical
transfection methods, the cell viability was low due to cell shearing or reagent toxicity whereas
chemical transfections, proved to be only transient with low efficiencies were achieved. The
data from this research shows a clear distinction between the large (18.5+kbp) pRTS-1 plasmid
and the small (3kbp) control GFP plasmid. When all other parameters are kept equal,
transfections with the control plasmid shows minimal disturbance to cell viability and high
transfection efficiency while pRTS-1 transfections presented massive cell death post-transfection.
Furthermore, large quantities of cells that survive transfection were lost upon selection.
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An alternative approach to resolving the low transfection efficiency issue is to re-
engineer the beneficial portions of the pRTS-1 vector (TET-repressor and the response/reporter
elements of TET-On) into viral vectors. If the benefits of removing super-physiological doses of
BMP-2 are higher than the risks, genetically engineering pRTS-1 into MSCs through viral
transfections may be considered. As reviewed by Thomas et al., adenovirus may package up to
30kbp depending on the helper agent but the high inflammatory potential makes it less desirable.
Alternatively, viral vectors such as retrovirus or lentivirus can package approximately 8kbp.111
The two enhancements of the pRTS-1 can be excised down to roughly 11kbp but this can be
reduced further if the reporter gene of the bidirectional promoter is excised (figure 5.5). This
would still allow a tight regulation while having both elements of the TET-On system
incorporated into a viral vector with low inflammatory potential.
The second experimental limitation was associated with the cell type. HUCPVC, which
has not been fully characterized, proved to be a difficult target for large plasmid transfections.
Figure 5.5 pRTS-1 Vector – The red area indicates the regions that include the response and
reporter element as well as the TET-repressor responsible for low background activity.
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HUCPVCs were unable to proliferate when a large plasmid was incorporated and as a result,
alternate cell types were used in this research thesis.
The third experimental limitation was the verification of the VEGF-A construct. The
purpose of this gene was to induce angiogenesis at the site of transplant. As explained in the
introduction, vascularization of transplanted tissue quickened the regeneration process.80
The
difficulty in this thesis was associated with the development of the pRTS-1-VEGF-A system.
Despite several attempts to confirm the orientation of the VEGF-A gene insert, the quality of the
sequencing data was always below the acceptable threshold. We carried on with cell
transfections to see if we can detect the expression at a protein level, however, we were unable to
detect VEGF-A from any clones.
The final experimental limitation was associated with RUNX2 and OSX constructs.
RUNX2 and OSX are key genes necessary during osteoblastogenesis. Literature indicates that in
the absence of these genes, osteoblasts are not formed and as a result, no bone is formed.8 The
mechanism and signaling pathway is yet to be fully understood but due to their importance in
osteoblastogenesis, we created the pRTS-1-RUNX2 and pRTS-1-OSX constructs to learn the
effects of up regulating the genes above physiological levels. We were able to control the
expression level but failed to detect any enhancement to osteogenesis. Unfortunately, the current
understanding of RUNX2 and OSX could not explain the lack of osteogenic effect found in our
research.
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Conclusions
Five conclusions can be drawn from this research.
1. Stable clones of RBMCs and ROS cells transfected with the pRTS-1-BMP-2 plasmids
can be generated by electroporation.
2. Stable clones can be induced and un-induced, in a dose-dependent manner, with
doxycycline.
3. Stable clones can re-achieve near-maximum gene expression within 72 hours using
500ng/mL doxycycline.
4. Xfect transfected RBMCs and ROS cells rapidly lose gene expression upon removal of
the selection pressure.
5. Amaxa Nucleofection, Clontech Xfect, Invitrogen Lipofectamine, and Qiagen SuperFect
did not generate stable clones.
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Future Directions
The aim of this research was to study a controlled one-vector expression system through
non-viral transfection methods with MSCs. This research work implies that by controlling the
quantity and duration of gene expression during bone regeneration, two issues of the current
treatment can be addressed: the burst release of rhBMP-2, and the need for super-physiological
dose. But as discussed in section 5.5, several aspects of this research warrant further study.
1. in vivo engraftment of pRTS-1-X stable clones into animal models for ectopic bone
formation followed by bone regeneration in non-union bone defects.
2. Optimization and increasing transfection efficiency for the large pRTS-1 vector.
3. In addition, understanding the minor differences found between transfection methods and
attempting to explain the low transfection efficiency associated with large vectors would
also warrant further research.
4. Finally, it may be possible to re-engineer the beneficial portions of the pRTS-1 vector
(TET-repressor and the response/reporter elements of TET-On) into a viral vector to
study the potential to regulate gene expression with low background activity.
Page 108
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