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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

(a.kee@unsw.edu.au)

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 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

4 9 14 20 33

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.

4 9 14 20 33

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

Use of genetically modified mice

•  KO/KI mice – Cystic fibrosis – Muscular dystrophies

•  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

Microfilaments – cell shape, motility, cytokinesis

Intermediate filaments – provide strength, compression resistance

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Microfilaments Intermediate Filaments

Microtubules

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.

cell shape cell adhesion cell motility vesicle transport endocytosis exocytosis golgi function cytokinesis membrane function

Organization of actin filaments within cells

Organisation Participation

Classes of actin binding proteins

side binding proteins

cross-linking protein (in cell cortex)

capping (end-blocking) protein

severing protein

motor proteins

nucleation protein

monomer- sequestering protein

actin monomers

actin filaments

Tropomyosin

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Classes of actin binding proteins

side binding proteins

cross-linking protein (in cell cortex)

capping (end-blocking) protein

severing protein

motor proteins

nucleation protein

monomer- sequestering protein

actin monomers

actin filaments

Tropomyosin Tropomyosin

Tropomyosin (Tm)

Ø filamentous protein

Ø Forms dimers

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

Tropomyosins Binds Along the Sides of ActinMicrofilaments

Actin

Tropomyosin (>40 isoforms)

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

Ø  Tm5NM1/2 knockout

Using knockout and transgenic mice to understand the function of Tm5NM1 in vivo

– Tm5NM1/2 eliminated in all tissues

Ø  hTm5NM1 transgenic (Tg) – Tm5NM1 overexpressed in most tissues

X γTm 1a 1b 4 3 2b 9d

6 b 9c 5

6 a 9a 9b 8 7

Tm5NM1 Tm5NM2

Beta-actin promotor Human Tm5NM1

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Phenotypes in the Tm5NM1/2 KO & hTm5NM1 Tg mouse

Parameter     Affected/Unaffected  

Organ  Weights  (Fat,  brain,  kidneys)   ↓ KO  ↑Tg  

Adipocyte  Proliferation   ↓  KO    ↑Tg  

Body  Weight   Unaffected  

Histopathology   Unaffected  

Food  Intake   Unaffected  

Food  Utilisation  &  Metabolism   Unaffected  

Glucose  Uptake/Insulin  Secretion   ?  KO    ↑Tg  

tg

wt

* *

* *

*

* P < 0.01

Glucose Tolerance Test

Glucose Injection

KO

WT

Compensation from other Tms may be responsible for lack of an effect on glucose

clearance in Tm5NM1 KO mice

Tm5NM1

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Glucose homeostasis - coordinated by many organs and tissues of the body

Adapted from: Rosen & Spiegelman, 2006

0.0

1.0

2.0

3.0

4.0

5.0

6.0

basal +insulin

umol/g/h

wt/wttg/tg

Insulin-stimulated glucose uptake is increased in Tm5NM1 transgenic mice

Glucose Uptake – adipose tissue P < 0.05

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)

From: Ramm & James, 2005

GLUT4

GLUT4

1 Insulin signalling

2 GLUT4 translocation

Actin has a critical role in this process

Sec8 Sec 5 Myrip

Wave(1.6)

Tethering

Myo1c

Docking  

Tethering  

Components of GLUT4 trafficking increased in Tg mice

Actin

Adapted from: Zaid et al., 2008

Data based on Western blotting

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wt tg

Myo1c

Sec 8

Western blots showing components of GLUT4 trafficking increased in Tg mice

From adipose tissue

Tm5NM1 can produce stable actin filaments

Stable microfilaments Higher actin filament turnover Shorter filaments

Gunning, Schevzov, Kee, Hardeman. Trend Cell

Biol 2005

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: a.kee@unsw.edu.au) 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.