transgenic animals : stable integration of exogenous dna in the genome

Transgenic animals:

•Stable integration of exogenous DNA in the

genome

•Transgene is transmitted as a mendelean

character

Françoise POIRIERInstitut Jacques Monod, CNRS 7592Paris

Why studying the mouse?

1. Relevance to human

– 60 106 years between mouse/human

– in mammals, transcription starts early in

development

– Importance of extraembryonic tissues

– Immunology

– Human diseases

– Behaviour

2. Elaborate genetics experiments are possible !

Simple mouse facts

• Gestation time: 19-21 days

• Litter size: 6-15 pups

• Generation time: 10 weeks (5 generations/year)

• Mouse genome: 3. 109 base pairs - 30 000 genes (same

as human)

20 chromosomes (23 in human)

Mouse genetics

Beginning of 21thmouse genome sequenceadvances of molecular genetics Genome wide phenotype driven screens

Beginning of 20th century collection of natural mutations

End of the 70sMolecular biology « classical » transgenics

1970-1990ES cells (blastocyst)Homologous recombination « targeted » transgenesis

1990-1995CRE/lox system conditional mutations

1980 «Classical transgenesis»: random integration of cloned DNA

day1

- Recovery of a 1 day old embryo

1980 «Classical transgenesis»: random integration of cloned DNA

day1

- Recovery of a 1 day old embryo

-Injection of cloned DNA

1980 «Classical transgenesis»: random integration of cloned DNA

day1

- Recovery of a 1 day old embryo

-Injection of cloned DNA

- Transfer in a pseudo pregnant female

1980 «Classical transgenesis»: random integration of cloned DNA

day1

- Recovery of a 1 day old embryo

-Injection of cloned DNA

- Transfer in a pseudo pregnant female

- newborn babies

1980 «Classical transgenesis»: random integration of cloned DNA

day1

- Recovery of a 1 day old embryo

-Injection of cloned DNA

- Transfer in a pseudo pregnant female

day1

- Recovery of a 1 day old embryo

-Injection of cloned DNA

- Transfer in a pseudo pregnant female

- newborn babies- newborn babies

- tail DNA

- PCR test: transgenic or not? 10% FREQUENCY

Gain of function mutations

Classical transgenesis(additive)

Gene hypothesis transgenicsGH growth hormone big mice

Sry sex determination gene (sterile) XX males

Ras oncogene tumors

Mouse genetics

Beginning of 20th century collection of natural mutations

End of the 70sMolecular biology « classical » transgenics

1970-1990ES cells (blastocyst)Homologous recombination « targeted » transgenesis

1989 Targeted mutagenesis(substitution)

Rare event

Cannot be done directly in mice

Indirect method

1989 Targeted mutagenesis(substitution)

Rare event

Cannot be done directly in mice

Indirect method

Genome rearrangement is done in ES cells

new mouse line

day1

- Recovery of a 1 day old embryo

- Injection of DNA

- Transfer in a pseudo pregnant female

- Recovery of a 3 day old embryo

- Injection of cells

- Transfer in a pseudo pregnant female day3

1989 Targeted mutagenesis: injection of selected ES cells

Growth of clones

attachment

Growth and differentiation

Light dissociation and seeding on feeder cells

Selection and illimited passages

« feeders »: Embryonic fibroblasts

(LIF)

15% fœtal calf serum2- mercaptoethanol

129 blastocyst

day 1.5

day 5.5day 4.5

day 2.5

Day 5

ES cells

day 2

day 3

day 4

day5

ES cells

ES cells

day2

day 5

Holding Pipet

Blastocyst(day 3.5)

ES cells (12 to 15)

Injection pipet

ES cell line

Chimeric mouseor not?

ES cell line

Chimeric mouseor not?

Donor blastocyst (black mouse)

Host blastocyst (white mouse)

Chimeric mouse

ES cell line

Donor blastocyst (black mouse)

Host blastocyst (white mouse)

somaticchimerism

somaticchimerism

germline chimerism?

ES cells

Chimeric mouse x white mouse

Donor blastocyst (black mouse)

Host blastocyst (white mouse)

Transgenic animals

Chimeric male

Transgenic animal

Chimeric male

1. An ES cell can give rise to a transgenic line

2. How can we modify an ES cell before injection?

Homologous recombination

Goal: MAKE NULL MUTATIONS

homologous recombination is the way to target an endogenous gene

• Very frequent event in yeast

•Very rare event in mouse : ratio of H.R. over R.I. (1/105)

•Need for a selection system

•Can only be done in tissue culture cells in vitro, not possible in vivo

target the gene of interest in ES cells and transmit it as a mutation in vivo

Engeneering a null mutation with a replacement vector(positive/negative selection)

1 2 3 4Wt allele

Engeneering a null mutation with a replacement vector(positive/negative selection)

1 2 3 4Wt allele

5 ’ homology 3 ’homology

Engeneering a null mutation with a replacement vector(positive/negative selection)

1 2 3 4Wt allele

3 4plasmid

5 ’ homology 3 ’homology

subcloning

Engeneering a null mutation with a replacement vector(positive/negative selection)

1 2 3 4Wt allele

3 4plasmid

5 ’ homology 3 ’homology

Hybridization + crossing over

Engeneering a null mutation with a replacement vector(positive/negative selection)

1 2 3 4Wt allele

3 4plasmid

Hybridization + crossing over

3 4targeted allele

Deletion of exons 1 and 2 = null mutation

Very rare event in mouse : ratio of H.R. over R.I. (1/105)

How can we select?

ATG

Engeneering a null mutation with a replacement vector(positive/negative selection)

1 2 3 4Wt allele

Resistance to antibiotic (neomycine)

sensitivityto gancyclovir

(analogue of thymidine)

NEO 3 4 TKplasmid

5 ’ homology 3 ’homology

Engeneering a null mutation with a replacement vector(positive/negative selection)

1 2 3 4Wt allele

Resistance to antibiotic (neomycine)

sensitivityto gancyclovir

(analogue of thymidine)

NEO 3 4 TKplasmid

5 ’ homology 3 ’homology

Engeneering a null mutation with a replacement vector(positive/negative selection)

1 2 3 4Wt allele

3 4targeted allele

ATG

resistant to NEO + resistant to gancyclovir

NEO

1. Random integration of the full plasmid

NEO 3 4 TKplasmid

NEO TK

Selection medium neo/gancyclovir

Sensitivity to gancyclovir« negative selection »

NEO+ TK-

NEO 3 4plasmid

NEO

2. Random integration of a truncated plasmid

Selection medium neo/gancyclovir

NEO+ TK-

1 2 3 4

NEO 3 4 TK

Wt allele

plasmid

5’homology 3’homology

3 4NEOTargeted allele

3. Homologous recombination

Selection medium neo/gancyclocir

In practice…..

1. Isolate of 129 Sv genomic locus2. Construct a replacement vector

- 5 to 10 Kb of homology- plan for a deletion (remove ATG if

possible)

3. Electroporate 2. 107 ES cells /15 microgr plasmid DNA4. Apply G418 gancyclovir selection (clones NEO+TK-)5. Pick, amplify, freeze clones6. Screen HR clones by PCR and confirm by Southern blot

analysisaverage rate = 5% HR clones (locus

dependent)7. Inject of HR cells in host blastocysts

NEO 3 4 TKplasmid

5’homology 3’homology

Hundreds of new mouse lines carrying null mutations!

25% embryonic lethality

10% lethality between 3 and 6 weeks of age

Majority survival, many mutations do not display any obvious phenotype

Nobel Price 2007Martin Evans, Oliver Smithies, Mario

Cappechi

And what about the predictions ?

Yes insulin-/- diabetes

And what about the predictions ?

Yes insulin-/- diabetes

Yes/No src-/- viable but osteopetrosis

And what about the predictions ?

Yes insulin-/- diabetes

Yes/No src-/- viable but osteopetrosis

No MyoD-/- survival (functional redundancy)

And what about the predictions ?

Yes insulin-/- diabetes

Yes/No src-/- viable but osteopetrosis

No MyoD-/- survival (functional redundancy)

HNF3b-/- lethal at day 8 ofembryogenesis gastrulation phenotype

And what about the predictions ?

Yes insulin-/- diabetes

Yes/No src-/- viable but osteopetrosis

No MyoD-/- survival (functional redundancy)

HNF3b-/- lethal at day 8 ofembryogenesis gastrulation phenotype

FosB-/- viable behaviour defect

FosB+/+ mother

FosB-/- mother

There are limitations to the positive/negative gene targeting strategy

Only null mutations are possible and yet….

- 200 genes are implicated in Drosophila eye development but

95% of them are embryonic lethal

- somatic mutations can occur in life (cancer)

goal = obtain conditional mutations (time, space)

Mouse genetics

Beginning of 20th century collection of natural mutations

End of the 70sMolecular biology « classical » transgenics

1970-1990ES cells (blastocyst)Homologous recombination « targeted » transgenesis

1990-1995CRE/lox system conditional mutations

CRE/loxP system

5’-ATAACTTCGTATA GCATACAT TATACGAAGTTAT-3’3’-TATTGAAGCATAT CGTATGTA ATATGCTTCAATA-5’

assymetric core

Structure of LoxP site (34 bases)

LoxP LoxP LoxP

CRE Recombinase

+

ex ex ex

« floxed » targeted gene

homologous recombination Classical transgenic mouse

X

Constitutive CRE expression under the control of a specific promoter

Floxed-gene Specific CRE expression

Space specific inactivation

ex ex ex

« floxed » targeted gene

homologous recombination Classical transgenic mouse

Constitutive CRE expression under the control of a specific promoter

Floxed-gene Specific CRE expression

Space specific inactivation

ex ex ex

« floxed » targeted gene

homologous recombination Classical transgenic mouse

Specific inactivation in CRE expressing cells

X

Constitutive CRE expression under the control of a specific promoter

Floxed-gene Specific CRE expression

(Ex: inactivation of HNF3 in liver cells)

Space specific inactivation

ex ex ex

Flox-BRCA1 PromWAP-CRE

« floxed » exon 7 of BRCA1

WAP: gene specific of adult mammary gland (milk protein)

Targeting of BRCA1 in mammary gland epithelium

X

Obtain conditional mutations by using CRE in vivoSpace specific inactivation

Obtain conditional mutations by using CRE in vivoTime specific inactivation

Inactive CRE Active CREcomposé X

Obtain conditional mutations by using CRE in vivoTime specific inactivation

Inactive CRE Active CREInjection du composé X à la souris

Souris normale Inactivation of a floxed gene at a given time

Agonist(tamoxyphen)

Active CREinactive CRE

Ligand binding domain (steroid receptor)

Obtain conditional mutations by using CRE in vivoTime specific inactivation

Agonist(tamoxyphen)

Active CREInactive CRE

Ligand binding domain (steroid receptor)

Obtain conditional mutations by using CRE in vivo

Ubiquitous promoter-inactive CRE

Time specific inactivation

Agonist(tamoxyphen)

Active CREInducible CRE

Ligand binding domain (steroid receptor)

Ubiquitous promoter-inactive CRE

Floxed gene

X

Time specific inactivation

Agonist(tamoxyphen)

Active CREInducible CRE

Ligand binding domain (steroid receptor)

Ubiquitous promoter-inactive CRE

Floxed gene

Double transgenic

X

Time specific inactivation

Floxed gene

Ubiquitous promoter-inactive CRE

X

Double transgenic

tamoxiphen

Agonist (tamoxyphen)

Active CREInducible CRE

Ligand binding domain (steroid receptor)

Time specific inactivation

Floxed gene

Ubiquitous promoter-inactive CRE

X

Double transgenic

Agonist (tamoxyphen)

Active CREInducible CRE

Ligand binding domain (steroid receptor)

Inactivation of the floxed gene at the time of injection

Obtain conditional mutations by using CRE in vivo

tamoxiphen

Time specific inactivation

Floxed gene

specific promoter-inactive CRE

X

Double transgenic

Agonist (tamoxyphen)

Active CREInducible CRE

Ligand binding domain (steroid receptor)

Inactivation of the floxed gene at the time of injection in CRE expressing cells

Obtain conditional mutations by using CRE in vivo

tamoxiphen

Time and space specific inactivation

Targeted mutations have brought a wealth of informationhttp://tbase.jax.-org

• Development

• Mouse models for several human diseases are available:Cardiovascular defectsSkeletal and growth defectsResponse to pain stimuliDeafnessMuscular dystrophy

1. «Reverse genetics»

from the gene to the phenotype 

Targeted mutagenesis = gene driven approach

many genes have subtle (unexpected) roles to play that may

become apparent only when sensitive (appropriate)

assays are used

2. «Forward genetics »

from the phenotype to the gene

Random mutagenesis = phenotype driven approach

a phenotypic screen gives an immediate link between a gene

and its function

History of modern mouse genetics 

Mouse genetics

Beginning of 21thmouse genome sequenceadvances of molecular genetics Genome wide phenotype driven screens

Beginning of 20th century collection of natural mutations

End of the 70sMolecular biology « classical » transgenics

1970-1990ES cells (blastocyst)Homologous recombination « targeted » transgenesis

1990-1995CRE/lox system conditional mutations

ENU= N-ethyl-N-nitrosourea

Ethylation of O or N in DNA

Point mutations44%A/T T/A38% A/T G/C

• Wide range of mutations : null, gain of function, hypomorph

64% missense10% non sense26% splicing errors

• Random effect: no bias

ENU mutagenesis: random point mutations

ENU: high efficiency mutagenesis

ENU treatment of males

Effective mutagenesis in the early spermatogonial cells

Mutated sperm produced during the entire life of the animal

1 mutation in a given gene occurs every 175-655 gametes screened

Systematic phenotype driven screens are now undertaken

Success depends on the establishment of a coordinated network of researchers involved in rapid non-invasive screening of every mutant mouse created.

1) Size, skeleton, skin, activity, ataxia

2) Semi quantitative tests: motor neurons, muscle, sensory functions

4) Metabolic parameters

3) Behaviour tests

….. but a single base is mutated out of 3x109 base pairs!!!

• integrated genetics and physical maps (mouse and human)

step1: backcross in order to define a physical interval for the mutation

• BAC transgenesis large stable genomic fragments are cloned in

bacterial artificial chromosomes (contigs)

step2: rescue the phenotype with a BAC

How about storing the mutants?Mouse genetics, particularly phenotype driven

screens, would not be possible without freezing/thawing of mouse lines

• mouse embryos can be frozen

• mouse sperm can be frozen

Strategy:

1) identify categories of mutants for every key phenotypic area of interest (developmental stages, immunology, behaviour, disease related, etc, etc…)

2) freeze down the mutants

C. Elegans RNAi (knock down)

Zebrafish morpholinos (knock down) the genome underwent duplications

Chicken no genetics

Xenopus morpholinos (knock down) the genome underwent duplications

Drosophila genetics, but no way to store mutants

Mouse powerful genetics, possibility of freezing lines it is a mammal … (but expensive and time consuming)

Functional genomics in various developmental systems