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Genetic Manipulation Of The Mouse
1. Genetic Manipulation
of the Mouse in
Developmental Cardiology
Fraz Ahmed Ismat, MD
Clinical Associate in Pediatrics
The Children’s Hospital of Philadelphia
2. Forward
Genetic
Screens
Embryo & Direct
Tissue Genetic
Manipulation Manipulation
Population
Analysis
3. fly mouse zebrafish
Forward
Genetic
frog mouse
Screens
Embryo & Direct
Tissue Genetic
Manipulation Manipulation chick
chick Population zebrafish
Analysis
human
5. Types of Genetic Manipulation
in Mouse
Transgenics Random
Insertion
Homologous Targeted
Recombination Modification
6. Reasons for Genetic Manipulation
in Mouse
1) Similarity to People (anatomically, anyway)
Global
2) Disruption of Normal Genes
Selective
3) “Lineage Tracing”
11. Transgenics in Mouse
Limitations
1) Limited knowledge of the promoter
(NFATc1 enhancer)
2) No regulation of insertion site
(CCS-LacZ)
3) What happens to daughter cells?
12. Homologous Recombination I
A B C D Genomic
Locus
Targeting
Insert
Vector
A′ B′ C′ D′
Recombined
Insert Locus
A B C D
13. Homologous Recombination II
Genomic
5′ arm 3′ arm
Locus
negative
5′ arm Insert neo 3′ arm tox selection
positive
selection
14. Homologous Recombination II
Genomic
5′ arm 3′ arm
Locus
negative
5′ arm Insert neo 3′ arm tox selection
positive
selection
no insertion
death
15. Homologous Recombination II
Genomic
5′ arm 3′ arm
Locus
negative
5′ arm Insert neo 3′ arm tox selection
positive
selection
random
insertion
no insertion
negative
selector
death
death
16. Homologous Recombination II
probe probe Genomic
5′ arm 3′ arm
Locus
negative
5′ arm Insert neo 3′ arm tox selection
positive
selection
random
insertion
no insertion targeted insertion
+neo R -tox negative
selector
death survival
death
18. From Target to Mouse
(in only a few years…)
vector
es cells
19. From Target to Mouse
(in only a few years…)
vector
es cells
blastocyst
targeted
cells
20. From Target to Mouse
(in only a few years…)
vector
es cells
blastocyst
targeted
cells
implanted into
surrogate
21. From Target to Mouse
(in only a few years…)
vector
es cells
blastocyst
targeted
cells
implanted into chimeric
wild type
surrogate offspring
22. From Target to Mouse
(in only a few years…)
vector
es cells
blastocyst
targeted
cells
germline!
implanted into chimeric
wild type
surrogate offspring
23. From Target to Mouse
(in only a few years…)
vector
es cells null!
blastocyst
targeted
cells
germline!
implanted into chimeric
wild type
surrogate offspring
24. Homologous Recombination III
What can the insertion consist of?
bar bar bar-null allele
bar mutant
bar (point mutation, dominant
negative, gain-of-function,…)
“foo” substitution
foo
(alternate gene, marker,…)
bar “floxed” bar
? what?
25. What is “floxed” for?
loxP site
“floxed” bar
bar
“flanked” by loxP sites (i.e. “floxed”)
In combination with Cre Recombinase…
CONDITIONAL KNOCKOUT!
29. “Lineage Tracing”
(foo-cre × Rosa-LacZ)
“foo” expression
Daughter cells express
LacZ with or without
“foo” expression
Tissue of
also expreses “foo”, interest
but outside of our
tissue of interest
30. “Lineage Tracing”
(foo-LacZ)
“foo” expression
Daughter cells only
express LacZ
concurrent with
Tissue of
“foo”
Still interest
expressing
“foo”
32. Current Problems
OVERLAP of two (or more) regions
(e.g. Neural crest & secondary heart field)
Reliability of current-generation reporters
(e.g. ROSA26 vs. GATA4-Flap)
TRUE lineage tracing