The Use of Genetically-Modified Mouse Models to Study the Cytoskeleton
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1 The Use of Genetically-Modified Mouse Models to Study the Cytoskeleton Anthony Kee (PhD) Neuromuscular and Regenerative Medicine Unit School of Medical Sciences 2012
2 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?
3 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
4 Creation of transgenic mice Fertilised oocyte Taken from: Strachen & Read Human Molecular Genetics
5 Transgenic mice random integration Types of transgenics 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.
6 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-tropomyosin slow (Met9Arg) Skeletal muscle specific Early stages of embryonic muscle Mutant protein causes Nemaline Myopathy
7 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.
8 Variable expression with random insertion of transgenes Ø The foreign DNA usually integrates as linear arrays, results in variable levels of gene dosage. wt Transgenic lines α-tm slow (Met9Arg) α-tm slow Nemaline Transgenic Mice
9 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. 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. wt Transgenic lines α-tm slow (Met9Arg) α-tm slow Nemaline Transgenic Mice
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11 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.
12 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].
13 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.
14 Homologous recombination is normal when germ cells are formed chromosome from mother chromosome from father Homologous recombination increases the genetic variability in germ cells
15 Making a knockout mouse requires 1. Pluripotent embryonic stem (ES) cells
16 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.
17 In vitro culturing of ES cells Pluripotent ES cell colonies Differentiated ES cell colony
18
19 Knockout- How? 4. Genetically modified ES cells injected into blastocyst 1. ES cells isolated from blastocyst 3.5dpc 3. Microinjection microscope use to take up ES cells with glass syringe 2. DNA targeting construct into the ES cells 5. Blastocyst implanted into surrogate mother
20 Making a knockout mouse requires 1. Pluripotent embryonic stem (ES) cells 2. Construction of KO vector by standard cloning procedures
21 Construction of KO/KI DNA vector Mouse genome * DNA vector carrying a mutation Standard molecular biology techniques are used to design and make the targeting DNA vector
22 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
23 Selecting for gene replacement events Ø Targeting vector contains marker genes Ø Positive selectable marker, neomycin phosphotransferase 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.
24 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
25 Positive and negative selection of recombinant ES cells Recombinants with random insertion Non recombinants Ganciclovir kills the recombinants with random insertion Treat with neomycin (positive selection) Treat with ganciclovir (negative selection) Recombinants with gene-targeted insertion
26 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
27 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
28 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
29 ES cells from a brown mouse Blastocyst from a white mouse
30 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
31 Screening of Mice +/+ -/- +/- Adapted from
32 Time-line for making a knockout mouse
33 KO/KI Versus Transgenic mice Gene targeting 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 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
34 Use of genetically modified mice KO/KI mice Cystic fibrosis Muscular dystrophies Transgenic mice To study dominantly acting alleles of mutated genes (nemaline myopathy)
35 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.
36 KO/KI Summary Transgenic
37 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.
38 Conditional Knockouts Cre Recombinase Approach Introns (insertion in noncoding region allows normal protein expression) Target Gene LoxP sites Transfect into ES cells in culture Blastocyst injection & breeding as per normal KO Target Gene X Tissue specific promoter Cre Target gene deleted in cells expressing Cre
39
40 The Cytoskeleton Microfilaments cell shape, motility, cytokinesis Intermediate filaments provide strength, compression resistance Microtubules organelle and vesicle transport, cell division
41 Microfilaments Intermediate Filaments Microtubules
42 Actin monomers (G actin) with the aid of actin binding proteins can polymerize to form actin filaments (F actin) Within cells an equilibrium exists between monomeric and filamentous actin. This equilibrium is influence by actin binding proteins.
43 Organization of actin filaments within cells Organisation Participation cell shape cell adhesion cell motility vesicle transport endocytosis exocytosis golgi function cytokinesis membrane function
44 nucleation protein actin monomers monomersequestering protein severing protein actin filaments cross-linking protein (in cell cortex) side binding proteins motor proteins Tropomyosin capping (end-blocking) protein Classes of actin binding proteins
45 nucleation protein actin monomers monomersequestering protein severing protein actin filaments cross-linking protein (in cell cortex) side binding proteins motor proteins Tropomyosin capping (end-blocking) protein Classes of actin binding proteins
46 Tropomyosin (Tm) Tropomyosin (>40 isoforms) Ø filamentous protein Ø Forms dimers Actin Ø Dimers interact head-to-tail to form a polymer Ø Tm polymer interacts along the length of the actin filament Ø Provides stability and regulates the binding of other actin binding protein to the actin filament
47 Different Tm isoforms regulate actin filaments by recruiting different actin binding proteins Stable microfilaments Higher actin filament turnover Shorter filaments Gunning, Schevzov, Kee, Hardeman. Trend Cell Biol 2005
48 Using knockout and transgenic mice to understand the function of Tm5NM1 in vivo γtm 1a 2b 1b a 6 b 7 89a 9b 9c 9d X Tm5NM1 Tm5NM2 Ø Tm5NM1/2 knockout Tm5NM1/2 eliminated in all tissues Beta-actin promotor Human Tm5NM1 Ø htm5nm1 transgenic (Tg) Tm5NM1 overexpressed in most tissues
49 Phenotypes in the Tm5NM1/2 KO & htm5nm1 Tg mouse Parameter Organ Weights (Fat, brain, kidneys) Adipocyte Proliferation Body Weight Histopathology Food Intake Food Utilisation & Metabolism Glucose Uptake/Insulin Secretion Affected/Unaffected KO Tg KO Tg Unaffected Unaffected Unaffected Unaffected? KO Tg
50 Glucose Tolerance Test wt * * * * * tg * P < 0.01 Glucose Injection
51 WT KO
52 Compensation from other Tms may be responsible for lack of an effect on glucose clearance in Tm5NM1 KO mice Tm5NM1
53 Glucose homeostasis - coordinated by many organs and tissues of the body Adapted from: Rosen & Spiegelman, 2006
54 Insulin-stimulated glucose uptake is increased in Tm5NM1 transgenic mice umol/g/h Glucose Uptake adipose tissue wt/wt tg/tg P < basal +insulin
55 The obligatory step in insulin-stimulated glucose uptake is translocation of the glucose transporter (GLUT4) to the plasma membrane Glucose Transport Pathway (muscle and fat) 1 Insulin signalling Actin has a critical role in this process GLUT4 GLUT4 From: Ramm & James, 2005
56 Components of GLUT4 trafficking increased in Tg mice Tethering Docking Tethering Sec8 Sec 5 Myrip Actin Myo1c Adapted from: Zaid et al., 2008 Wave(1.6) Data based on Western blotting
57 Western blots showing components of GLUT4 trafficking increased in Tg mice From adipose tissue wt tg Myo1c Sec 8
58 Tm5NM1 can produce stable actin filaments Stable microfilaments Higher actin filament turnover Shorter filaments Gunning, Schevzov, Kee, Hardeman. Trend Cell Biol 2005
59 Can Tm5NM1 increase in actin filaments in the mouse tissues? Tm5NM1
60 Tm5NM1 increases filamentous actin in adipose tissue F-actin staining
61 Mechanisms: Tm5NM1 and Glucose Uptake Wild-Type Mice Exocyst Sec8 Sec5 Myrip VAMP2 Syntaxin4 Synip Munc18c Glucose Mobilisa(on Myo1c Ac?n Docking Fusion GLUT4 Tethering Adapted from Zaid et al., 2008
62 Mechanisms: Tm5NM1 and Glucose Uptake Tm5NM1 Transgenic Mice Mobilisa(on GLUT4 Exocyst Myo1c Sec8 Sec5 Myrip Tethering Tm5NM1 Longer more stable unbranched filaments Docking Syntaxin4 Synip Munc18c Fusion Glucose Adapted from Zaid et al., 2008 Note: indicate upregulated in Tm5NM1 Tg mice
63 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
64 Looking for keen Honours Students! Supervisors: Dr. Anthony Kee & Professors Edna Hardeman & Peter Gunning (contact: 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.
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