[II] Molecular Techniques for Studying Gene Expression

[II] Molecular Techniques for Studying Gene Expression

Basics of recombinant DNA technology Methods used to monitor the expression of genes RT-PCR vs. Real-time RT-PCR DNA microarray analysis Transgenic analysis and gene inactivation Chromatin immunoprecipiotation Methods of analysis of proteins Reading List II

Cloning and Characterizing DNA Molecule **What is a recombinant DNA molecule? **Cloning of a genomic gene **Cloning of a complementary DNA (cDNA)

Why is it necessary to clone genes?

• Naturally occurring DNA molecules are very long and a single molecule usually carrying many genes

• Genes may occupy only a small proportion of the chromosomal DNA and the rest are noncoding sequences (a human gene might constitute only 1/1,000,000 of a chromosomal DNA)

• Unlike enzymes, there is no convenient method to identify a gene. Therefore, it is impossible to obtain a sizable amount of DNA encoding a gene by the ordinary purification method

•

• It is impossible to purify individual mRNA due to lacking method to identify individual mRNA species. Clone individual cDNA of the mRNA is the only solution

Technological Breakthrough in Molecular Biology Leading to Cloning of DNA

• Discovery of restriction enzymes & modification enzymes --- allowing cutting and manipulation of DNA molecules

• Methods of isolating DNA molecules and agarose gel electrophoresis for separating DNA molecules

• Plasmids and bacteriophage as vectors for in vivo amplification of DNA molecules

• Methods of introducing DNA molecules into cells --- “Transformation”

• Hybridization method for identifying specific DNA molecules

• Reading List: Restriction enzyme – a background paper Recombinant DNA and gene cloning

General Strategy of Recombinant DNA Technology

• Recombinant DNA technology: cloning and manipulation of DNA molecules (genomic DNA or cDNA)

• Vector + DNA fragment Recombinant DNA

Replication of recombinant DNA molecules in host cells

Isolation, sequencing, and manipulation of purified DNA fragment

Several Enzymes that Digest DNA Molecule

• (a): Terminal phosphatase

• (b): Nuclease: digest either the first ester bond or the second ester bond

• (c): Endonuclease

• (d): Exonuclease

Restriction Enzyme (I)

• Restriction enzymes were originally discovered in bacterial cells serving to remove invading foreign DNA, but bacterial DNA itself can be protected from restriction enzyme digestion by adding methyl group (-CH3) to A or C on the DNA

• Restriction enzymes were discovered by W. Arber, D. Nathans and H. Smith in 1960’s. They were awarded with a Nobel Prize in 1978

• DNA fragments generated by digestion with some restriction enzymes are with sticky ends

• Restriction enzymes have been used to prepare DNA molecules for cloning

• Reading Assignment:

Nobel lecture by H. Smith 1978

Restriction Enzymes (II)• Restriction enzymes: Enzymes from bacterial cells that can cut

DNA molecules at specific nucleotide sequence

Restriction site (palindromic sequence)

• Some enzymes digest DNA to produce sticky ends while others produce blunt ends

• Palindromic sequence; 4 cutters = 44 (256 bases); 6 cutters = 46

(4096 bases)

Restriction Enzymes (III)

(a). Some restriction endonucleases that cleave the restriction sites to generate a staggered cut

(b). Other restriction endonucleases that cleave the restriction sites to generate blunt ends

Restriction Map of a DNA Fragment

• A restriction map of a DNA fragment is a linear sequence of sites separated by defined distances on DNA

• The map shown above identifies three restriction sites cleaved by restriction enzyme A and two sites by restriction enzyme B

• Thus DNA fragment digested by restriction enzyme A alone generates 4 fragments and digested by restriction enzyme B alone generates 3 fragments

• These DNA fragments can be resolved by electrophoresis on an agarose gel and their determined

DNA Modification Enzymes• DNA Modification Enzymes: Enzymes that can modify DNA

molecules DNA Ligase: An enzyme that can ligate two DNA molecules together

by making a phosphodiester bond DNA polymerase I: involves in synthesis of DNA molecules (identified

by Athur Kormberg) DNA phosphorylase: An enzyme that can remove phosphate group

from DNA molecules DNA kinase: An enzyme that can add a phosphate group onto the 5’-

end of a DNA molecule Terminal transferase: An enzyme that can add nucleotide on to 3’-end

of the DNA molecule Endo- and exo-nucleases: break a phosphodiester bond Reverse transcriptase: make a strand of DNA by copying RNA

template (identified by H. Temin and D. Baltimore independently) Tqa polymerase: synthesize DNA under high temperature. This

enzyme is isolated from Thermus aquatica (identified by T.D. Brock)

Cloning Vectors (I): Basic Components of a Bacterial Plasmid Cloning Vector

• Plasmid cloning vector in E. coli is a circular DNA molecule of 1.2 to 3 kb

• Plasmid Vectors contain: Selectable gene

such as ampr

Replication origin (ORI)

A synthetic polylinker with unique restriction enzyme recognition site

• DNA fragment from a few base pairs to about 10 kb can be inserted into the cloning vector and manipulated

Construction of a Recombinant

Plasmid

• Replication origin

• Selection marker: Ampicillin resistant gene or others

• MCS: mutiple cloning site

• DNA fragment of interest can be inserted at MCS for amplification

• DNA fragment smaller than 10 Kb can be amplified in a bacterial plasmid

Cloning Vectors (II): Cloning Vectors for Cloning Genomic DNA (the earliest version)

1. A Phage Cloning Vector: Lambda () phage DNA has been developed into a cloning vector for cloning DNA fragment up to 20 kb. This is the earliest type of vector used to construct a genomic library

Recombinant Phage

Cloning Vectors (III): Cosmid and Phagemid• Cosmid is a type of hybrid plasmid (often used as a cloning

vector) that contains cos sequence. Cosmids (cos sites + plasmid = cosmid) DNA sequences are originally from the Lambda phage. Cosmids can be used to build genomic library.

• A phagemid is developed as a hybrid of the filamentus phage M13 and plasmid to produce a vector that can grow as a plasmid, and also be packaged as single stranded DNA in viral particles. It contains an ORI for double stranded replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles.

Cloning Vectors (IV): Cloning vectors for cloning a large piece of DNA

• Bacterial artificial chromosome (BAC): A plasmid contains a

functional fertility factor, replication origin, partition factor, selection factor and T7 and Sp6 promoters

This plasmid (F-plasmid) can accommodate DNA fragment of about 100-300 Kb

The recombinant plasmid can be introduced into E. coli cells by electroporation

• Yeast Artificial Chromosome:

This cloning vector contains Tel (telomere), CEN (centromere) and ORI (replication origin) of yeast cells. It can accept a large piece of DNA (100 kb to 3,000 kb)

The recombinant cloning vector is introduced into yeast cells by electroporation for amplification

Cloning Vectors (IV): Cloning vectors for cloning a large piece of DNA

Cloning Vectors for Different Purposes

• Detection of Nucleic Acids Agarose gel electroporation Hybridization, Southern blot and RNA

northern blot analyses

Agarose Gel Electrophoresis to Separate DNA

• Agarose is an inert carbohydrate isolated from seaweeds. DNA molecules of different sizes can be separated in an agarose gel by electrophoresis and visualized by staining the gel with ethedium bromide & observe under UV light. The DNA fragment can be recovered from the gel by extraction with phenol and chloroform

DNA Fragments Visualized Under UV Light

DNA on agarose gel is stained with ethedium bromide and observed under UV light

Sizes of DNA Fragments Determined by Agarose Eectroporation

Separation of Molecules by Gradient Centrifugation

• Macromolecules (DNA, RNA and proteins) can be separated by their sizes or densities

• Macromolecules of different sizes can be separated by electrophoresis on agarose or polyacrylamid gels

• Macromolecules of different densities can be separated by grant centrifugation Sedementation

centrifugation Equilibrium

centrifugation

Hybridization• Hybridization: Two fragments of single-stranded

homologous DNA molecules can form hybrid through hydrogen-bonding formation (base pairing)

5’-GTACTTAGGCAATTGGGCA-3’ 3’-CATGAATCCGTTAACCCGT-5’

• If one of these two strands of DNA is labeled with radioactive isotopes, the hybrid will be easily visualized by autoradiography

• Hybridization can occur between two homologous DNA molecules or a DNA molecule and a RNA molecule

• “Hybridization” has been used in Southern blot and RNA northern blot analyses

Southern Blot Hybridization

This method is developed by Edwin Southern

Results of Southern Blot Hybridization

Fluoerescence in situ Hybridization

This technique is used for cytological localization of molecules in the cell

Methods of Introducing DNA into Cells

• Recombinant DNA molecules can be transferred into bacterial cells, plant cells and animal cells by transformation methods:

Bacterial cells: regular transformation method or electroporation method

Plant cells: Agrobacterium infection, electroporation or particle gum bombardment

Animal cells: microinjection, Ca3(PO4)2 precipitation method, lipofection or electroporation method

Electroporators

Electroporators can generate high frequency pulses to create transient opening on cells by which the DNA molecules enter into the cell

• Cloning of Genomic DNA and cDNA

Complementary (c) DNA Verses Genomic Gene

Complementary DNA (cDNA): A reverse sequence of an mRNA, synthesized by using mRNA as a template and reverse transcriptase as the enzyme to catalyze the reaction

Genomic Gene: A gene contains the coding region (both intron and exon) and the control region

Why should one clone a cDNA or a genomic gene?

Purposes of Cloning Genomic DNA and cDNA

• Genomic DNA: Studying structures of genes, gene families Identifying promoters and other regulatory elements Studying evolution of genes

• cDNA: Studying structures of mRNA Measuring the levels of mRNA Studying developmental stage-specific and tissue-specific

expression of genes Studying processing and stability of mRNA Producing recombinant proteins

Construction of Genomic DNA Library

• Since the sizes of genomic genes vary from several kbs to several hundred kbs, bacterial plasmid vector is not very suitable for cloning a complete gene

• Commonly used vectors for cloning complete genes are: Lambda phage vector (up to 20-25 kb) Artificial bacterial chromosome (BAC) vector (up to 500 kb): it

contains a bacterial origin of DNA replication and genes required to regulate their own replication

Yeast artificial chromosome (YAC) vector (up to 1000 kb): it contains all the elements of yeast chromosome i.e., elements for replication of the chromosome during S phase, segregation of chromosome, telomeres, genes for selection and DNA fragment to be cloned

• Genomic DNA library: A collection of DNA fragments of a genome

Isolation of Genomic Genes from DNA Libraries

• Isolating gene clones from libraries by colony hybridization or plaque hybridization

• Characterizing the inserted DNA by: Restriction enzyme digestion (establishing restriction

maps) Determining the nucleotide sequence of the inserted DNA Expressing the gene product in E. coli or mammalian

cells

Colony Hybridization

• This method is based on the principle that two homologous strands of nucleic acids can form hybrid form

• If one of the strands of nucleic acid is radio-labeled, the hybrid can be visualized by autoradiography

Plaque Hybridization

• This technique is suitable for isolating genes cloned in a lambda phage cloning library

Restriction Enzyme Mapping

• Restriction enzyme recognition sites can be used to make maps of DNA --- restriction mapping

• Restriction map of a cloned DNA is made by digesting the cloned DNA with a series of restriction enzymes and determine their sites on the DNA

Restriction map of the catfish growth hormone gene

Structure of Dideoxynucleotide Triphosphate

• The nucleotide sequence of a piece of DNA can be determined by: Chemical sequencing

method developed by Maxam Gilbert

Dideoxy chain-termination method developed by F. Sanger

• Incorporation of dideoxynucleotide triphosphate into the going chain of DNA will stop the elongation of the DNA chain

A Single Strand DNA to be Sequenced

Assigned Reading: Nobel lecture by F. Sanger on “DNA sequencing”, 1980

Separating the Products on a Denaturing Polyacrylamid Gel

3’

Separating the Products on an Automated Sequencing Machine

(1.8 x 106)

(1.2 x 107)

(9.7 x 107)

(1.0 x 108)

(1.8 x 108)

(3.2 x 109)

Strategies of Assembling Whole Genome Sequence

Chromosome Walking

• This technique allows the isolation of a long eukaryotic gene

• An alternative is to construct a BAC library that contains long piece DNA molecules

Making cDNA from an

Eukaryotic Gene

• To express an eukaryotic gene into its product in bacterial cells, the mRNA is converted into cDNA

• The cloned cDNA can be expressed into protein product by inserting it into a plasmid containing a functional promoter (expression vector)

Construction of a cDNA Library

• Synthesis of 1st strand cDNA

• Synthesis of double strand cDNA

• Ligate the double stranded cDNA into a plasmid vector orphage vector

• Propagate the library in E. coli cells

Lambda Phage Cloning Vector for Cloning cDNA

1. Phage Cloning Vector:Lambda () phage DNA has also been developed for cloning cDNA as well. This type of vector is suitable for cloning cDNA banks. In this case, the non-essential region in the lambda phage genome is not removed. If a functional promoter is present in the mutiple cloning site, the cloned cDNA will also express into it gene product

cDNA Library (Bank)

• Lambda gt10: for cloning regular cDNA that can be identified by hybridization

• Lambda gt11: for

cloning cDNA that can express the protein product, so the cDNA clone can be identified by immunoblotting

Expression Vectors

• Expression vector: A cloning vector that contains a “functional promoter”, if this vector also contains a gene that its expression can be easily seen (detected), it is said that this vector contains a “reporter gene”

Expression of a lacZ gene stained for -galactosidase in a mouse embryo

Leuciferase gene derived from fireflies is a popular reporter gene. An expression vector containing leuciferase gene can be used to define the functional promoter and other regulatory sequence

• External and internal images of bone metastasis of AC3488 GFP

• (A) External images of tumors in the skeletal system [including the skull (arrow heads), scapula (thick arrows), spine (fine arrows), and liver metastasis (largest arrows) in a dorsal view of live, intact nude mouse

• (B–I) Series of external fluorescence images of metastatic lesions in the skull, ribs, spine, and tibia, (B, D, F, and H) compared with corresponding images of the exposed skeletal metastases (C, E, G, and I) (Bars =1280 m).

Visualization of Cancer Cell Growth by GFP

• Methods used to monitor the expression of genes

Using Cloned DNA Fragments to Study Gene Expression RNA northern blot analysis In situ hybridization RNase protection assay

Measuring mRNA levels by RT-PCR or real-time RT-PCR assay

Measuring the profiles of gene expression by DNA microarray

Measuring the profiles of gene expression by RNA sequencing analysis

Using Cloned DNA Fragments to Study Gene Expression

• Hybridization techniques permit detection of specific DNA fragments and mRNAs Southern blotting method: DNA-DNA hybridization RNA northern blotting method: RNA-DNA hybridization,

qualitative and semi-quantitative methods In situ hybridization RT-PCR

• DNA microarrays can be used to evaluate the expression of many genes at one time

• E. coli expression systems can produce large quantities of proteins from cloned genes

• Cloned genes can also be introduced into yeast cells and other animal cells for production of large quantities of proteins for analysis

DNA Southern Blot & RNA Northern Blot Techniques

RNA

RNA Northern Blot Analysis of -Globin mRNA

• Total RNA was extracted from erythrolukemia cells grown but not induced and in cells induced to stop growing and allowed to differentiate for 48 h or 96 h

• The RNA samples were resolved in agarose gels and transferred to a nylon membrane

• The membrane was hybridized to radio-labeled -globin cDNA

In Situ Hybridization to Detect of the mRNA of Sonic Hedgehog in Mouse Embryos

Whole embryo (10 days old)

A section of the embryo

Drosophila Embryo

In this experiment, the in situ hybridization technique is used to localize the site of expression of sonic hedgehog gene in mouse embryos

Principle of RNase Protection Assay

AAAAAAAmRNA

32P DNA

+

Hybridization and digested with RNase

The product is analyzed on a urea-polyaccrylamid gel

RNase Protection Assay to Measure IGF-I mRNA Levels in the Liver of Rainbow Trout

If a standard curve is established using known quantities of cRNA, the amount of the unknown can be determined

g cRNA

CP

M o

r O

D60

0

Nuclear Run-on Transcription Assay

• Nuclear run-on assay can be used to ascertain which gene is active in a given cell allowing transcription to continue in isolated nuclei

• Specific transcript can be identified by their hybridization

to known DNAs on dot blot • It can also be used to determine

the effects of assay conditions on nuclear transcription

• Transcription activity of a specific gene can be determined

• It can also be used to measure template activity

Nuclear Run-On Assay to Measure the Transcription Rates of Genes in Various Tissues

Measuring levels of mRNA by RT-PCR or real-time RT-PCR assay

Principle of Reverse Transcriptioin (RT)-

PCR• End Point RT-PCR:

Measuring the presence of a particular mRNA

Can be adapted to perform semi-quantitative determination of an mRNA

• Real-time RT-PCR: For quantitative measurement of the level of an mRNA Absolute quantitative RT-

PCR Comparative quantitative

RT-PCR

PCR Amplification Program• 1 cycle at 95 oC for 3 min to denature all templates

• 40-50 cycles: 95 oC for 15 sec for denaturation of PCR products At annealing temperature for 15 sec to anneal primers

to the templates 72 oC for 30 sec for synthesis of DNA

Determine Levels of mRNA by Semi-quantitative RT-PCR

• If levels of -actin mRNA in various tissues are constant, then the levels of mRNA of interest can be expressed in terms of relative to levels of -actin

• This assumption will only work when the increase of levels of mRNA is in the linear range of the detection

• Example given in the left of this slide is showing how the levels of NPY mRNA is determined by this method

Competitive Quantitative RT-PCR

In vitro transcription

Competitor mRNA

AAAA

IGF-II mRNA to be determined

ReverseTranscription

ReverseTranscription

Competitor cDNA IGF-II cDNA

Various amounts of both templates and fixed amount of amplification primers and proceed PCR amplification

Competitive Quantitative RT-PCR

When the ratio of target/competitor equals to 1, the levels of competitor equals the level of the target

Assigned Reading: Greene and Chen, 1999. Quantitation of IGF-I, IGF-II, and multiple insulin receptor mRNAs during embryonic development in rainbow trout. Molecular Reproductive Development 54: 348-361.

Real-time PCR Machine

Principle of Real-Time RT-PCR

• SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to DNA. The resulting DNA-dye-complex absorbs blue light (λmax = 497 nm) and emits green light (λmax = 520 nm). The stain preferentially binds to double-stranded DNA, but will stain single-stranded DNA with lower performance. SYBR green can also stain RNA with a lower performance than DNA.

• Since double stranded DNA can interact quantitatively with SYBR Green 1 preferentially and thus can be used to monitor the amount of DNA synthesized after each cycle of PCR reaction.

• Cycle threshold (CT): CT is defined as the number of PCR cycles required for the fluoescent signal to cross the threshold (i.e., exceeds background level). This is usually determined automatically by the real-time PCR machine set in the factory

Determination of Cycle Threshold (CT)

    1 2 3 4 5 6 7 8 9 10 11 12

A

Content                       

Sample                        

Peak 1                        

Peak 2                       

B

Content   Unkn-1 Unkn-1 Unkn-1 Unkn-5 Unkn-5 Unkn-5 Unkn-9 Unkn-9 Unkn-9   

Sample                        

Peak 1   82.20 82.00 82.00 82.20 82.00 82.00 82.00 82.00 82.00    

Peak 2   None None None None None None None None None   

C

Content   Unkn-2 Unkn-2 Unkn-2 Unkn-6 Unkn-6 Unkn-6 Unkn-10 Unkn-10 Unkn-10   

Sample                        

Peak 1   82.20 82.00 82.00 82.00 82.00 82.00 82.00 82.00 82.00    

Peak 2   None None None None None None None None None   

D

Content   Unkn-3 Unkn-3 Unkn-3 Unkn-7 Unkn-7 Unkn-7 Unkn-11 Unkn-11 Unkn-11   

Sample                        

Peak 1   82.20 82.00 82.00 82.00 82.00 82.00 82.00 82.00 82.00    

Peak 2   None None None None None None None None None   

E

Content   Unkn-4 Unkn-4 Unkn-4 Unkn-8 Unkn-8 Unkn-8 Unkn-12 Unkn-12 Unkn-12   

Sample                        

Peak 1   82.20 82.00 82.00 82.00 82.00 82.00 82.00 82.00 82.00    

Peak 2   None None None None None None None None None   

F

Content   NTC-1 NTC-1 NTC-1               

Sample                        

Peak 1   None None None                

Peak 2   None None None               

G

Content                       

Sample                        

Peak 1                        

Peak 2                       

H

Content                       

Sample                        

Peak 1                        

Peak 2                       

Sample Set Up in a 96-Well Plate

PCR Amplification Program

• 1 cycle at 95 oC for 3 min to denature all templates

• 40-50 cycles: 95 oC for 15 sec for denaturation of PCR products At annealing temperature for 15 sec to anneal primers to

the templates 72 oC for 30 sec for synthesis of DNA

• From the melting profile, one can determine whether the PCR product is amplified from a single target

Melting Profile of RT-PCR Products

Absolute Quantitative Real-time RT-PCR

• Construction of a standard curve: Construct a synthetic cRNA of your interest Reverse transcribe the cRNA into cDNA Set up Real-time PCR with known and increasing amount of

cDNA Plot Ct value vs. starting quantity of cDNA

Levels of Gene Expression Determined by Absolute RT-PCR

NKX2.5 GATA 5

Norm. GI,GII

β-actin

Norm. GI,GII Norm. GI,GII

Norm.

GI,GII

Norm.

GI,GII

Norm.

GI,GII

Assigned Reading: Chun et al., 2006. Trout Ea4- or human Eb-peptide of pro-IGF-I disrupts heart, red blood cell, and vasculature development in zebrafish embryos

Levels of Gene Expression Determined by Absolute RT-PCR

GATA 1 GATA 2 VEGF

Norm. GI,GII

Norm.

GI,GII

Norm. GI,GII

Norm.

GI,GII

Norm. GI,GII

Norm.

GI,GII

Reduction of mRNA Levels of Heart, Vasculature and Blood Cell Development-Related Genes in rtEa4-Peptide Injected Embryos

Gene

Molecules of mRNA/embryo at 36 hpf

NormalDefective embryos

Fold of reduction

NKX2.5 2.77±2.04X106 8.02±5.96X103 242.1±1.0

GATA5 3.94±1.07X105 2.41±6.58X104 11.7±0.9

GATA1 1.63±0.46X106 2.27±0.12X105 5.5±1.2

GATA2 1.37±0.22X104 3.59±0.76X103 2.7±1.0

VEGF 2.87±0.96X105 3.09±0.41X104 6.4±1.1

β-actin 5.77±1.19X106 4.16±1.33X106 1.0±1 .0

Relative Quantitative RT-PCR

Fold =1/[2-(∆ ∆ CT)]

∆CT= CT (target) – CT (normalizer)

∆∆CT = ∆CT (treated) - ∆CT (control)

Calculation of Relative Expression Levels

Asigned Reading: Chen et al., 2007. Suppression of growth and cancer-induced angiogenesis of aggressive human breast cancercells (MDA-MB-231) on the chorioallantoic membraneof developing chicken embryos by Epeptide of pro-IGF-I

GAPDH: glyceraldehyde-3-phosphate dehydrogenase or -actin mRNA is usually used as normalizer

Comparative Real-Time PCR Analysis of Genes Up- and Down-Regulated by E-Peptide

Names of Genes Relative Expression Levels

uPA 0.52 + 0.10PAI 1 0.42 + 0.04BCL 2 0.28 + 0.04Cysteine-rich angiogenesis inducer 0.86 + 0.12Tumor-associated Ca++ signal inducer 0.44 + 0.12TYPO3 protein tyrosine kinase 3.02 + 0.80Tumor rejection antigen (Gp96) 3.46 + 0.96Heat shock protein 90 KDa protein 13.40 + 0.35Heat shock 70 KDa protein 10 2.92 + 0.39Capase 3 4.58 + 0.35Fibronectin 1 2.32 + 0.31Laminin receptor 1 1.41 + 0.11

*Fold of reduction = 2-(∆ ∆ CT), where ∆ ∆ CT is ∆ CT sample - ∆CT control where ∆ CT = CT (Target) – CT (normalizer)

• Microarray: a technique that allows you to determine the expression of many genes at one time

Why DNA Microarray Technology?

• Gene discovery: Examples Profiling of cancer-specific expressing genes Tissue-specific expression of genes Developmental-specific expression of genes

• Disease diagnosis: Collections of genes showing expression of genes specific

to certain types of diseases Examples: Specific cancer type, hematopoietic disease etc.

• Drug discovery: Pharmacogenomics To find correlations between therapeutic responses to drug

and gene profiles of patience Toxicological research: Toxicogenomics• To find correlations between toxic responses to toxicants

and changes in the gene profiles of the objects exposed to such toxicants

Principle of DNA Microarray

• Genes (cDNA or oligonucleotides are spotted on glass plates

• Messenger RNA is reverse transcribed into cDNA and labelled with Cy3 (emission 570nm, green) or Cy5 (emission 670 nm, red)

• Hybridization (mixing Cy3-cDNA and Cy5 cDNA)

• Scan the slide to detect the hybridization signals

Enlarged Photo of a Microarray Chip

• This array has 2400 human genes

• Red indicates increase of expression; yellow equal expression and green reduce of expression

• This technique can help to determine the profiles of gene expression

Interpreting the Scanned Image

• The measured intensities from the two fluorescent reporters have been false-colored red and green and overlaid

• Yellow spots have roughly equal amounts of bound cDNA from each cell population and so have equal intensity in the red and green channels (red + green = yellow)

• Spots whose mRNA’s are present at a higher level in one or the other cell population show up as predominantly red or green

• The ratio of fluorescent intensities for a spot is interpreted as the ratio of concentrations for its corresponding mRNA in the two cell populations

Example

Novel Anti-cancer Activity of Pro-IGF-I E-Peptide

Assigned Reading:Chen et al., 2007: Suppression of Growth and Cancer-Induced Angiogenesis of Aggressive Human Breast CancerCells (MDA-MB-231) on the Chorioallantoic Membraneof Developing Chicken Embryos by E-peptide of Pro-IGF-I

B C A DS E

Signal peptide

Mature IGF-I E-peptide

Insulin-Like Growth Factor (IGF) I

Post-translational processing

EB C A DS

Primary translation product of IGF-I

E-Peptide of Pro-IGF-I

• The pro-hormone peptide of IGF-I

• It contains about 77 amino acid residues

• Presents in fish, human and other animals

• It is highly soluble in aquaous buffers

• Can be readily prepared by expressing the gene in E. coli and other cell systems

• Exerts dose-dependent mitogenic activities in established non-transformed cell lines (Tian et al., 1999)

• Induces morphological differentiation and inhibits anchorage-independent growth in oncogenic transformed cell lines (Chen et al., 2002; Kuo and Chen, 2002)

• Inhibits tumor cell growth and invasion, and tumor-induced angiogenesis in developing chicken embryos (Chen et al., 2007)

• Induces programmed cell death in cancer cells (Chen et al., 2011)

• Up-regulate fironectin 1 and laminin receptor genes and down-regulate uPA, tPA and TIMP1 (Siri and Chen 2006a, 2006b)

Novel Anti-Cancer Activities of the E-Peptide

Is There Molecular Evidenced

• If E-peptide of IGF-I can control cancer cell growth, there must be genes that are up- or down-regulated by E-peptide

• Using microarray analysis technique, we determined genes related to cancer cell activities that are up- or down regulated by e-peptide in aggressive human breast cancer cell (MBA-MD-231) Isolate RNA samples from MBA-MD-231 cells treated wit E-

peptide and untreated cells Prepare cDNA from both RNA smples Labele E-peptide treated cDNA with Cy3 and the untreated

cDN with Cy5 Combine both labeled cDNA and hybridized to a high

density human gene chip and scan the results

Results of the Microarray Assay

• From repeat screening a gene chip contain 10,000 human genes, more than 1,000 genes were found to be up- or down-regulated by E-peptide to a different degree

• Those genes with ratio of treated/untreated signals over + or – 2 are considered real difference

• Examples of up-regulated and down-regulated genes are summarized in the next two slides

• To confirm the microarray results, the mRNA levels of each gene in the treated and untreated RNA samples should be determined by real-time quantitative or comparative RT-PCR

Some Genes Down-Regulated by E-Peptide

• Oncogenes: TC 21, P53, vav 1, v-myb, c-H-ras (p21), sarc, v-erb 2 (high expression in tumors)

• Angiogenesis: Cysteine-rich angiogenic inducer 61 IGF-II, IGF-IIR, IGFBP-3, -4 & -7

• Cell adhesion: Catenin -like-1, Integrin -3, Integrin -3, Integrin -4, Tight junction protein 1

• Proteases: Cathepsin Z, Plasminogen activator inhibitor type I, Urokinase plasminogen activator, Tissue plasminogen activator

• Cell cycle, growth & proliferation: Cyclin-independent kinase inhibitor 1A, Latent transforming growth factor -binding protein 3, Serine inducible kinase, Colony stimulating factor 1, Thymosin -4

• Cytoskeleton molecules: Keritin-7, -8, and -18

Some Genes Up-Regulated by E-Peptide

• Cell adhesion: Fibronectin 1, Laminin receptor 1

• Cytoskeleton molecules: Keratin-1, Restin

• Proteases and inhibitors: Tissue inhibitor of metalloprotease I, Cathepsin C, Zinc metalloprotease

• Cell cycle, growth & proliferation: Cyclin A2, B1, B2, Cyclin-dependent kinase 7, Topoisomerase II

• Cell signal transduction molecules: TyrO3 protein kinase, Mitogen-activated protein kinase kinase 3, c-sar tyrosine kinase, Phosphotidylinositol-4-phosphate- 5 kinase type I

• Tumor rejection antigen (gp96) 1, Cdc 14 phosphatase, Protein tyrosine kinases, Heat shock proteins 70 and 90

Comparative Real-Time PCR Analysis of Genes Up- and Down-Regulated by E-Peptide

Names of Genes Relative Expression Levels

uPA 0.52 + 0.10PAI 1 0.42 + 0.04BCL 2 0.28 + 0.04Cysteine-rich angiogenesis inducer 0.86 + 0.12Tumor-associated Ca++ signal inducer 0.44 + 0.12TYPO3 protein tyrosine kinase 3.02 + 0.80Tumor rejection antigen (Gp96) 3.46 + 0.96Heat shock protein 90 KDa protein 13.40 + 0.35Heat shock 70 KDa pr5otein 10 2.92 + 0.39Capase 3 4.58 + 0.35Fibronectin 1 2.32 + 0.31Laminin receptor 1 1.41 + 0.11

RNA-Seq TechnologyRNA-seq, also called "Whole Transcriptome Shotgun Sequencing" ("WTSS"), refers to the use of high-throughput technologies to sequence cDNA or RNA in order to get information about a sample's RNA content. The technique has been adopted in studies of diseases like cancers. With deep coverage and base-level resolution, this technology provides information on differential expression of genes, including gene alleles and differently spliced transcripts; non-coding RNAs; post-transcriptional mutations or editing; and gene fusions

Reading Assignment:

1. RNA-Seq

2. Wang et al., 2009. RNA-Seq: a revolutionary tool forTranscriptomics. Nature Review: Genetics 10: 57-63.

• Transgenic technology Biotechnological application

Assigned Reading :Application of recombinant DNA technology

Gene Transfer in Plants

Transfer genes that can make vitamin A into rice

Golden Rice

Transgenic Animal Technology

• Transgenic Animals: Animals with foreign gene(s) integrated into their genomes

• The first transgenic animal was produced in mice by Richard Palmiter and Ralf Brinster in early 1980s

Transgene: Human growth hormone gene driven by the promoter of metallothionein gene

Procedure for Producing Transgenic Mice

• We have been working on gene transfer technology since mid 1980s

• Over the past two decades, we have produced several speccies of transgenic aquatic animals by microinjection, electroporation, infection with pantropic retroviral vectors, and lipofection

• References: Zhang et al., MRD 25: 3-13, 1990. Lu et al., Mol Marine Biol. Biotech 1:366-

375, 1991. Lu et al., PNAS 93: 3482-3486. 1996. Lu et al., Mol Marine Biol Biotecnnol 7:

289-295, 1997. Sarmasik et al., Marine Biotechnol 3: 465-

473, 2001 Sarmasik et al., Marine Biotechnol 3: S117-

S184, 2002 Lu et al., Marine Biotechnol 4: 328-337,

2002.

Transgenic Aquatic Animals

Pore Formation on Bacterial Cell Membrane by Cecropin

Cecropin Pores Formation

Prototype of Transgene

CMVPromoter

IgG-SP Cecropin P1

Transfer the transgene construct into sperm of rainbow trout by electroporation

Sperm-Mediated Gene Transfer

Transgene

Parameters: Dilution buffer, Dilution factor, DNA/sperm, Voltage, Pulse #

Families % Mortality (Mean ± S.D.)

F2 Generation F3 Generation

Mortalities of F2 and F3 Cecropin P1 Transgenic Fish Challenged with A. salmonicida

S8#Y419S7#375S9#746S7#342S8#505S9#638S9#659U6#768

35 ± 4

18 ± 8

26 ± 814 ± 840 ± 10

30 ± 5

79 ± 12

15 ± 512 ± 314 ± 520 ± 630 ± 320 ± 410 ± 225 ± 680 ± 3

Non-transgenic 80 ± 6 85 ± 8

For each family, challenge was conducted with 50 fish/family (0.4 to 0.5 g body weight) in triplicates and the dose of A. salmonicida used in each challenge study brings about 80% mortality in non-transgenic fish.

Some Key Terms

• Totipotent: Fertilized oocytes and blastomeres of 2, 4-, and 8-cell stage embryos. Could contribute to the development of the embryo proper and the placental tissue.

• Pluripotent: Inner cell mass (ICM) of blastocysts, embryonic ectoderm and primordial germ cells of fetal stages. Could contribute to the development of embryo proper only.

• Embryonic germ cells: Stem cells developed from primordial germ cells. Could develop into all somatic cell types of an embryo. These cells are also pluripotent.

• Somatic stem cell: Stem cells could only develop into restricted cell types. (e.g. Skin)

Stem cells: Stem cells are cells found in most, if not all, multi-cellular organisms. They are characterized by the ability to renew themselves through mitotic cell division and differentiation into a diverse range of specialized cell types.

Two types of mammalian stem cells are:

Embryonic stem cells: Isolated from the inner cell mass of blastocyst

Adult stem cells: Found in adult tissues.

Assigned Reading: Embryonic stem cells.

Totipotent and Pluripotent Cells

Different Paths of Generating Human ES Cells from In Vitro Fertilized Human Oocytes

Inactivation of Genes by Knock-Out Method

• Gene knock-out: Interrupt a gene by inserting a small piece DNA into it

• To perform this technique, it requires: ES or EG cells Introduce the interrupting DNA into ES or EG cells and

isolate recombinants Injecting the recombinant cells into blastoceal cavity of

mouse embryos Transfer the embryos into pseudo-pregnant female

mice

Assigned Reading: Elimination of a gene by the knock out technology

Construction of a Gene-Targeted Disruption Construct

• neor: a marker gene that confers resistant to G-418

• tkHSV: a marker gene confers sensitivity to ganciclovir

• When the above construct is introduced into mouse stem cells, stem cells with gene been disrupted can be isolated

• The isolated ES cells can be used to generate gene knock out animals

Isolation of Recombinant Stem Cells with a Gene Been Interrupted

Production of Germ Line Knock Out Animals

• Introduction of knock out heterozygous ES cells into the blastoceal cavity of mouse embryos

• Transfer embryos into pseudopregnant female mice

• Select chimeric mice• Mat chimeric mouse with homozygous

black mouse• Select brown mice for homozygous knock

out mice

Knock Out Genes in Somatic Cells by the loxP-Cre Recombination System

• loxP site: site-specific DNA recombina-tion site

• Cre: enzyme catalyzing recombina-tion

Inactivation of a Gene by Using RNAi

• In vitro or in vivo production of siRNA

• Inhibition of mex3 mRNA by injecting siRNA into the cells

• Methods of analyzing proteins produced in different tissues or composition of proteins in different cells:

SDS-polyacrylamide gel electroporation Immuno western blot analysis Two dimensional electroporation analysis Proteomic analysis

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9 Silver staining

Anti Histag Antibody

25.9kd19.4kd14.8kd

6.0kd

25.9kd19.4kd14.8kd

6.0kd

25.9kd19.4kd14.8kd

6.0kd

1 2 3 4 5 6 7 8 9

SDS Polyacrylamide gel stained with coomassie brilliant blue dye

SDS polyacrylamide gel stained with silver stain

Immuno western blot analysis

Immuno Western Blot Analysis

Concept of Two Dimensional Gel Electrophoresis

Comparison of Two Protein Samples

Strategy of Sequencing a Protein by Mass-Spectrometry

Identification of Proteins in Organelles by Gradient Centrifugation and LC-MS/MS Analysis

Chromatin Immunoprecipitation

This technique can be used to detect protein-DNA interactions in the native chromatin context in vivo. The associated DNA is purified for analysis by identifying its specific sequence by PCR or by labeling the DNA and applying to a tiling array to detect genome wide interactions

Assigned Reading [II]

1. Restriction Enzymes: a background paper2. Recombinant DNA and gene cloning3. Nobel lecture by H. Smith, 19784. Nobel lecture by O. Shimomura on discovery of GFP5. Nobel lecture by K. Mullis PCR, 19936. Nobel lecture by Sanger7. Nobel lecture by Michael Smith8. Elimination of a gene by the knock out technique9. Greene and Chen, 1999. Quantitation of IGF-I, IGF-II……..10. Chun et al., 2006: Trout Ea4 and human Eb-peptide ……..11. Chen et al., 200712. Embryonic stem cells13. RNA-Seq14. RNA-Seq: a revolutionary tool for transcriptomics