transgenic animals : stable integration of exogenous dna in the genome
DESCRIPTION
Françoise POIRIER Institut Jacques Monod, CNRS 7592 Paris. Transgenic animals : Stable integration of exogenous DNA in the genome Transgene is transmitted as a mendelean character. Why studying the mouse ?. 1. Relevance to human 60 10 6 years between mouse/human - PowerPoint PPT PresentationTRANSCRIPT
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