1 The Use of Genetically-Modified Mouse Models to Study the Cytoskeleton Anthony Kee (PhD) Neuromuscular and Regenerative Medicine Unit School of Medical Sciences ([email protected]) 2012 Why Study Gene Function • How does development happen? • Understand the function of gene products in adults • Genetic disease - when something goes wrong, where, when and how? Approaches to manipulate the mouse genome 1) Transgenic Random chromosomal integration of foreign DNA 2) Homologous Recombination Site directed disruption ( knockout ) or replacement with a modified variant of a gene allele (knock-in). Conditional KO/KI – tissue specific and inducible Creation of transgenic mice Taken from: Strachen & Read Human Molecular Genetics Fertilised oocyte
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Why Study Gene Function - CellBiology · (negative selection) Recombinants with random insertion Recombinants with gene-targeted insertion Non recombinants Ganciclovir kills the ...
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The Use of Genetically-Modified Mouse Models to Study the
Cytoskeleton
Anthony Kee (PhD)
Neuromuscular and Regenerative Medicine Unit School of Medical Sciences
• Understand the function of gene products in adults
• Genetic disease - when something goes wrong, where, when and how?
Approaches to manipulate the mouse genome
1) Transgenic Ø Random chromosomal integration of foreign DNA
2) Homologous Recombination Ø Site directed disruption (knockout) or
replacement with a modified variant of a gene allele (knock-in).
Ø Conditional KO/KI – tissue specific and inducible
Creation of transgenic mice
Taken from: Strachen & Read Human Molecular Genetics
Fertilised oocyte
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Transgenic mice – random integration
1) Target protein may be overexpressed: excessive amounts of normal protein expressed in tissues which normally express it, eg GH transgenic.
2) Target protein may be ectopically expressed: protein expressed in tissues which do not normally express it.
3) Mutated protein is expressed to produce: constitutively active (“gain-of-function”) or dominant negative (“loss-of-function”) form of a protein or to mimic a mutated protein observed in a human genetic disease.
Types of transgenics
Context-specific expression of the transgene
Design transgenic construct that contain transcriptional regulatory elements that direct expression to a specific cell type or developmental stage.
Example: Transgenic mouse model of human muscle disease Human skeletal actin promotor Human α-tropomyosinslow(Met9Arg)
Skeletal muscle specific
Early stages of embryonic muscle
Mutant protein causes
Nemaline Myopathy
Issues with random insertion of transgenes
Ø Because of the random nature of the transgene insertion, each resultant founder contains the transgene in a different site in the genome.
Ø The position effect can profoundly affect the expression of both the transgene and the endogenous genes whose regulatory elements may be disrupted.
Ø Transgene may disrupt endogenous genes (insertional mutagenesis) confounding the phenotype.
Variable expression with random insertion of transgenes
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α-Tmslow (Met9Arg)
α-Tmslow
Transgenic lines wt
Nemaline Transgenic Mice
Ø The foreign DNA usually integrates as linear arrays, results in variable levels of gene dosage.
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Transgenic lines Ø Therefore, it is essential that lines from several
different founder lines be examined before a conclusion relating a specific phenotype to transgene expression is made.
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α-Tmslow (Met9Arg)
α-Tmslow
Transgenic lines
wt
Nemaline Transgenic Mice
Ø To assess dose-response relationships between transgene expression and phenotype, it is also important to assess lines of mice which express the transgene at different levels.
What is a KO/KI mouse
Ø A mouse in which a specific mouse gene has been genetically modified and the modification is transmitted through the germ-line.
Ø KO (knockout) is a modification in which the activity of the gene is eliminated (eg. delete the gene or a key region)
Ø KI (knockin) is a modification in which a specific mutation(s) or rearrangement is introduced and the gene remains functional.
Why make a KO/KI mouse Ø Create mouse models to study pathophysiology of
disease and test therapeutic approaches to disease.
Ø Most useful to mimic recessive disorders (loss of function mutations).
Ø [Traditional transgenics can be used for dominant disorders].
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How to make a KO mouse • Principle is homologous recombination
Ø A fragment of genomic DNA is introduced into a mammalian cell and it can locate and recombine with the endogenous homologous sequences. This type of homologous recombination is also commonly refer to as gene targeting.
Ø It occurs in yeast, bacteria and certain viruses however it is a rare event in mammalian cells except germ cells.
Ø Transfected DNA most commonly integrates into a random chromosomal site.
Ø The relative frequency of targeted to random integration events will determine the success of generating a KO mouse.
Homologous recombination is normal when germ cells are formed
Homologous recombination increases the genetic variability in germ cells
chromosome from father
chromosome from mother
Making a knockout mouse requires
1. Pluripotent embryonic stem (ES) cells
ES cells can differentiate into all the different types of cells in the body
Ø ES cells are isolated from the Inner Cell Mass of a 3.5 day old mouse embryo.
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In vitro culturing of ES cells
Pluripotent ES cell colonies Differentiated ES cell colony
Knockout- How? 4. Genetically modified ES cells injected into blastocyst
1. ES cells isolated from blastocyst 3.5dpc
2. DNA targeting construct into the ES cells
5. Blastocyst implanted into surrogate mother
3. Microinjection microscope use to take up ES cells with glass syringe
Making a knockout mouse requires
1. Pluripotent embryonic stem (ES) cells
2. Construction of KO vector by standard cloning procedures
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Mouse genome
DNA vector carrying a mutation
Construction of KO/KI DNA vector
*
Standard molecular biology techniques are used to design and make the targeting DNA vector
Making a knockout mouse requires
1) Pluripotent embryonic stem (ES) cells
2) Construction of KO vector by standard cloning procedures
3) Introducing the KO vector into the ES cells by electroporation
4) Selecting for gene targeting events
Selecting for gene replacement events
Ø Targeting vector contains marker genes
Ø Positive selectable marker, neomycin phospho-transferase is resistant to the antibiotic neomycin.
Ø Negative selectable marker, thymidine kinase from Herpes Simplex virus. The TK gene confers sensitivity to the chemical gancyclovir.
ES cells with this construct will grow in culture medium containing neomycin but will not survive in the presence of ganciclovir. Resistant to neomycin and sensitive to ganciclovir
ES cells with this construct will grow in culture medium containing neomycin and ganciclovir. Resistant to neomycin and ganciclovir
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Positive and negative selection of recombinant ES cells
Treat with neomycin (positive selection)
Treat with ganciclovir (negative selection)
Recombinants with random insertion
Recombinants with gene-targeted insertion
Non recombinants Ganciclovir kills the recombinants with random insertion
Making a knockout mouse requires
1) Pluripotent embryonic stem (ES) cells
2) Construction of KO vector by standard cloning procedures
3) Introducing the KO vector into the ES cells by electroporation
4) Selecting for gene targeting events
5) Screening the ES colonies for the inserted DNA
Screening the ES colonies
• To identify which ES cells accepted the KO gene
DNA is isolated from the ES cells, cut with restriction enzymes, run on a gel and hybridised with radioactively labelled DNA probes. This is to test for the organisation of the target gene.
Example of Southern blot
+/+ +/+ +/- +/+ +/+
Making a knockout mouse requires
1) Pluripotent embryonic stem (ES) cells
2) Construction of KO vector by standard cloning procedures
3) Introducing the KO vector into the ES cells by electroporation
4) Selecting for gene targeting events
5) Screening the ES colonies for the inserted DNA
6) Injecting ES cells into the blastocysts
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ES cells from a brown mouse
Blastocyst from a white mouse Making a knockout mouse
requires 6) Injecting ES cells into the blastocysts
Ø The progeny will be a chimera consisting of both KO and wild type cells
Ø Hopefully some KO cells will contribute to the germ line.
Ø Heterozygous and homozygous progeny for the KO construct can be generated and analysed for phenotypic alterations
Adapted from http://nobelprize.org
Screening of Mice
+/+ -/-
+/-
Time-line for making a knockout mouse
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KO/KI Versus Transgenic mice
Advantage: Ø Specific insertion of gene at specific location or removal of specific gene
(knockout).
Ø Mimic recessive disorders (loss of function mutations).
Disadvantage: Ø Low level of ES cells with wanted gene inserted
Ø Further breeding necessary to obtain non-chimeric homozygotic animal
Gene targeting
Transgenic mice Advantage: Ø Relative high rate of insertion of the injected gene into the genome. Ø Use for dominant disorders.
Disadvantage: Ø Random insertion- can lead to position effects
• Transgenic mice – To study dominantly acting alleles of
mutated genes (nemaline myopathy)
Summary • Gene targeting is use to delete or mutate an
existing gene: KO and KI. Mice are generated by the injection into a blastocyst of genetically modified ES cells. Chimeric mice are made.
• Transgenic mice are use to study overexpression of a gene product. Mice are generated by DNA microinjection of fertilized oocytes. Results in random integration of the DNA.
• Both offer a valuable tool for the study of human disorders. ie. Dystrophies.
Transgenic
KO/KI Summary
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Advances in gene targeting • Ability to inactivate a gene at a specific time and in a
specific tissue. • Conditional gene targeting is achieved with the use of
the Cre/lox system. Ø Cre recombinase is an enzyme the catalyses
sequence specific recombination between two 34 base pair repeats (LoxP sites).
Ø The result of this recombination is deletion of the DNA between the LoxP sites.
Conditional Knockouts Cre Recombinase Approach
Target Gene
LoxP sites
Transfect into ES cells in culture
Blastocyst injection & breeding as per normal KO
Cre Target Gene Tissue specific promoter X
Target gene deleted in cells expressing Cre
Introns (insertion in non-coding region allows n o r m a l p r o t e i n expression)
The Cytoskeleton
Microtubules – organelle and vesicle transport, cell division
Can Tm5NM1 increase in actin filaments in the mouse tissues?
Tm5NM1
Tm5NM1 increases filamentous actin in adipose tissue
F-actin staining
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GLUT4
Microtubules
Mobilisa(on
Tethering
Docking Fusion
Syntaxin4 VAMP2 Synip
Munc18c
AcBn
Sec8 Sec5 Myrip
Exocyst
Myo1c
Glucose
Adapted from Zaid et al., 2008
Cytoskeletal
Remodelling
Mechanisms: Tm5NM1 and Glucose Uptake
Wild-Type Mice
Adapted from Zaid et al., 2008
Note: ↑ indicate upregulated in Tm5NM1 Tg mice
GLUT4↑
Microtubules
Mobilisa(on
Tethering
Docking Fusion
Syntaxin4 ↑ Synip ↑
Munc18c ↑
Sec8 ↑ Sec5 ↑ Myrip ↑
Exocyst
Tm5NM1 ↑ Myo1c ↑
Glucose ↑
Cytoskel
etal
Remode
lling
Longer more stable
unbranched filaments
Mechanisms: Tm5NM1 and Glucose Uptake
Tm5NM1 Transgenic Mice
Key points from the lecture
Overexpression of Tm isoforms achieved by the generation of genetically modified mice allow us to conclude that:
Ø Tm isoforms regulate actin filament function in vivo
Ø Tm5NM1 specifically regulates glucose uptake via altering the ratio of G- to F-actin
Looking for keen Honours Students!
Supervisors: Dr. Anthony Kee & Professors Edna Hardeman & Peter Gunning (contact: [email protected]) Project 1: Can altering the actin cytoskeleton improve insulin sensitivity in Type II diabetes? Use animals obesity models of Type II diabetes. Project 2: The role of tropomyosin in regulating glucose transport via its control of the actin cytoskeleton. Use cell culture models to understand molecule mechanisms.