cre-lox basics - generating knockout mice webinar...
TRANSCRIPT
Cre-lox Basics -
Generating
Knockout Mice
Technical Information Services
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Presentation Overview
Basic Cre-lox mechanism
Strain types and breeding schemes
o Tissue-specific knockouts
o General knockouts
o Inducible knockouts
o Reporters
Cre-lox web resources (finding mice, Cre
activity data, and more)
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A Revolutionary Genetic Tool
Cre-lox system
Natural part of P1
bacteriophage viral life cycle
Viral DNA injected into bacteria,
circularized using Cre-lox, and
replicated for development of
new viruses
Cre
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Cre-lox Successfully Engineered
in Other Organisms
Organisms
Yeast
Plants
Mammalian cell cultures
Mice
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Cre-lox Successfully Engineered
in Other Organisms
Organisms
Yeast
Plants
Mammalian cell cultures
Mice
Allows
Alteration & deletion of DNA
Regulation of location and
timing of gene recombination
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A Simple, Two Component System
Cre recombinase
Site-specific enzyme, catalyzes recombination
between two LoxP sites
loxP site
34 base pair DNA sequence
Location and orientation determines recombination result:
o Deletion o Inversion o Translocation
Reviewed in: Nagy A. 2000. Genesis 26(2):99-109. PMID:10686599
ATAACTTCGTATA-NNNTANNN-TATACGAAGTTAT
Abundant possibilities for genome manipulation!
Cre
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Mechanism: Cre-lox Deletion
Floxed target gene
Knockout allele
X
Ge
ne
X
LoxP
GeneX
LoxP LoxP GeneX
LoxP
Cre excision
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Mechanism: Cre-lox Inversion
Cre
LoxP LoxP GeneX
LoxP LoxP
GeneX
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Mechanism: Cre-lox Translocation
Cre
Chr 3
Chr 6 LoxP
LoxP
Reciprocal
Translocation (3;6) LoxP
LoxP
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Cre-lox Tissue-Specific Knockout
LoxP GeneX LoxP
GeneX LoxP LoxP
Homozygous “floxed” mouse
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Alb cre
GeneX
GeneX x
Liver-specific cre transgene Ex: B6.Cg-Tg(Alb-Cre)21Mgn/J
(003574)
Cre-lox Tissue-Specific Knockout
LoxP GeneX LoxP
Alb cre
GeneX
Cre-Lox mouse:
Heterozygous for gene X conditional knockout after 1 generation
Liver-specific cre transgene Ex: B6.Cg-Tg(Alb-Cre)21Mgn/J
(003574)
Alb cre
GeneX
GeneX
LoxP GeneX LoxP
GeneX LoxP LoxP x
Homozygous “floxed” mouse
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Cre-lox Tissue-Specific Knockout
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LoxP GeneX LoxP
Alb
GeneX
cre
Cre-lox Tissue-Specific Knockout (continued)
LoxP GeneX LoxP
GeneX LoxP LoxP
Hemizygous Alb-cre heterozygous “floxed” gene
25% homozygous for gene X conditional knockout (2nd generation)
GeneX
Alb cre
GeneX
LoxP LoxP
LoxP LoxP
x
Homozygous “floxed” mouse
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LoxP GeneX LoxP
Alb cre
GeneX
Cre-lox Tissue-Specific Knockout (continued)
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Alb-cre +
loxP + loxP +
Alb cre
Gene X
GeneX
Hemizygous
cre
Gene X
GeneX
loxP
Cre-lox Tissue-Specific Knockout (continued)
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Alb-cre +
loxP + loxP +
loxP Alb-cre
loxP/loxP
Alb-cre
loxP/+
+
loxP/loxP
+
loxP/+
Alb cre
Gene X
GeneX
Gene X
GeneX
Alb cre
Gene X
GeneX
25% homozygous for gene X conditional knockout (2nd generation)
Hemizygous
cre
Hemizygous Alb-cre heterozygous “floxed” gene
LoxP GeneX LoxP
GeneX LoxP LoxP x
Homozygous “floxed” mouse
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Alb cre
GeneX
LoxP GeneX LoxP
Improving Conditional Knockout Efficiency
Improving Conditional Knockout Efficiency
x
Heterozygous null mouse
(traditional knockout)
GeneX
Alb cre
GeneX
LoxP GeneX LoxP
12.5% conditional gene X knockouts
LoxP GeneX LoxP
Alb cre
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Hemizygous Alb-cre heterozygous “floxed” gene
Cre-lox Tissue-Specific Knockout (continued)
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Alb-cre +
loxP + loxP +
+
-
Alb cre
Gene X
GeneX
Hemizygous
cre
GeneX
Cre-lox Tissue-Specific Knockout (continued)
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Alb-cre +
loxP + loxP +
+ Alb-cre
loxP / +
Alb-cre
+ / +
+
loxP /+
+
+ / +
- Alb-cre
loxP / -
Alb-cre
+ / -
+
loxP / -
+
+ / -
Alb cre
Gene X
GeneX
Hemizygous
cre
GeneX
Alb cre
GeneX
12.5% conditional gene X knockouts
Improving Conditional Knockout Efficiency
Hemizygous Alb-cre heterozygous null mouse
x GeneX LoxP LoxP
Alb cre
GeneX
LoxP GeneX LoxP
LoxP GeneX LoxP
Alb cre
25% conditional gene X knockouts
Homozygous “floxed” mouse
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x Homozygous
“floxed” mouse
oocyte-specific cre expression
Ex: C57BL/6-Tg(Zp3-cre)93Knw/J
(003651)
ZP3 cre
Cre-lox Germline Knockouts
GeneX
LoxP GeneX LoxP
Ovary
ZP3 cre
Oocytes
2 more generations to produce
homozygote null mouse 26
Homozygous LoxP mouse Cre mouse – cre transgene (Tg)
early, widespread expression promoter
B6.Cg-(Sox2-cre)1Amc/J
(008454)
LoxP GeneX LoxP
LoxP GeneX LoxP Cre
GeneX
GeneX
Sox2
Cre-lox Knockout Breeding Scheme
Offspring: 50% heterozygous knockout after 1 generation
GeneX
x
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Offspring 2nd generation: 25% homozygous knockout
GeneX GeneX
x
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Cre-lox Knockout Breeding Scheme (continued)
Cre-lox Summary
Tissue-specific deletion
2 generations of breeding
Cre required to maintain line for
future generations
Genotype of whole mouse:
homozygous flox; Cre
Tissue-specific genotype:
homozygous flox-deleted; Cre
Germline/Embryonic deletion
2-3 generations of breeding
Cre not required after germline
deletion (can breed it out)
Genotype of whole mouse,
germplasm, organs & tissues:
homozygous flox-deleted
(knockout) for gene of interest
GeneX
LoxP GeneX
Cre Alb
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LoxP
LoxP
LoxP
Inducible Cre Mouse Models
x Homozygous LoxP mouse Inducible Cre mouse –
tamoxifen dependent Cre function
Ex: B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J
(004682)
Induce homozygous knockout of gene X with tamoxifen
+ Tamoxifen
2 Generations
LoxP GeneX LoxP
LoxP GeneX LoxP
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The FLP-FRT System
Analogous to the Cre-lox system
Derived from the yeast Saccharomyces cerevisiae
Becoming more popular in mouse-based research
Flipase recombinase (FLP) recognizes a pair of target
sequences (FRT) that flank a genomic region of
interest
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Using FLP-FRT with Cre-lox
LoxP LoxP PGK-neo exon exon FRT FRT
FLPase
LoxP LoxP exon exon FRT exon
exon
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Cre Considerations Mosaicism
Some target cells may not express Cre, or LoxP
sites may not recombine
May be integration site specific; evaluate multiple
cre transgenic founders
Expression in unexpected tissues (including germline)
Use Cre-positive males for breeding
Test expression using reporter
LoxP site recombination efficiency
Affected by position and distance
Heffner et al. 2012. Nat Commun 3:1218. PMID: 23169059
Schmidt-Supprian and Rajewsky. 2007. Nat Immunol 8(7):665-8. PMID:17579640
Cre mosaicism in
mouse mammary
gland epithelial
cells
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Cre Considerations (continued)
Breeding efficiency
If possible, have cre transgene on a different chromosome
than the floxed allele
Often good idea to breed out cre transgene after germline
deletion
Cre may produce a phenotype by itself
Insertion site effects; changes in gene expression
“Cre toxicity”
Target gene may be expressed prior to Cre recombination
Consider that genetic background may affect phenotype
Heffner et al. 2012. Nat Commun 3:1218. PMID: 23169059
Schmidt-Supprian and Rajewsky. 2007. Nat Immunol 8(7):665-8. PMID:17579640
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JAX Cre Reporter Strains
Livet J et al. 2007. Nature 450(7166):56-62. PMID:17972876
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Cre Reporter Strains
“Lox-Stop-Lox” (LSL)
Used to assess Cre activity in tissue(s) of interest
Only one generation of breeding needed
Ex: B6.129S4Gt(ROSA)26Sortm1Sor/J (003474)
Promotor LoxP LoxP lacZ
X
Promotor LoxP lacZ
STOP
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Cre Reporter Data
LacZ expression
following
widespread Cre
recombination
Soriano P. 1999. Nat Genet 21(1):70-1. PMID:9916792
Control
B6.129S4Gt(ROSA)26Sortm1Sor/J (003474)
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Cre Reporter Breeding Scheme
x B6.129S4Gt(ROSA)26Sortm1Sor/J
(003474) Cre reporter strain
Ex: nervous system-specific cre
B6.Cg-Tg(Nes-cre)1kln/J (003771)
**Could be any Cre recombinase strain
Cre
LacZ stain confirms Cre activity in expected tissues
LoxP LoxP LacZ
LoxP LoxP LacZ
Nestin
1 Generation
Cre Nestin
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Cre-lox Disease Model
Tuveson DA et al. 2004. Cancer Cell 5(4):375-87. PMID:15093544
B6.129S4-Krastm4Tyj/J (008179)
Promoter LoxP LoxP
Kras G12D
oncogene STOP
X
Promoter LoxP Kras G12D
oncogene
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Cre-lox Disease Model
x MMTV Cre
Only one round of breeding needed
Ex: STOCK Tg(MMTV-cre)4Mam/J
(003553)
LoxP STOP Kras G12D LoxP
LoxP Kras G12D
Mice develop
mammary neoplasms
B6.129S4-Krastm4Tyj/J (008179)
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JAX Cre Repository*
Largest collection of Cre-lox strains
580+ cre-expressing
200+ inducible Cre strains
1000+ floxed genes
210+ floxed-stop (LSL) Cre reporters
> 200 neuronal specific Cre strains
http://cre.jax.org/NeuroCres.html
http://cre.jax.org
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*as of Dec 2015
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JAX Cre Repository https://www.jax.org/research-and-faculty/tools/cre-repository
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JAX Cre Repository https://www.jax.org/research-and-faculty/tools/cre-repository
Searching for Cre Strains
https://www.jax.org/research-and-faculty/tools/cre-repository 44
muscle
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Searching for Cre Strains
JAX Cre Repository https://www.jax.org/research-and-faculty/tools/cre-repository
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JAX Cre Repository https://www.jax.org/research-and-faculty/tools/cre-repository
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JAX Repository: Cre Expression Data
B6.129S4-Gt(ROSA)26Sortm1Sor/J (003474)
Bred to LacZ reporter Strain 20X Lung
10X Heart
20X Skin 48
Evaluated @ four time points: E10.5, E15.5, P7 and
P56 (adult) o whole-mount or fresh frozen sections
o full necropsy (P7 & P56) – 11 organ systems, 30
organs/structures, 89 substructures
NanoZoomer slide scanner to capture data at 20X
Data shared with MGI Cre portal database
20X Lung
10X Heart
20X Skin
JAX Repository: Cre Expression Data
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Cre Portal @ MGI
www.creportal.org
Search by Site of Expression
or
Promoter
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muscle
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Cre Portal Search Results
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Cre Portal Search Results Link to Phenotypic Data, Images, & References
Cre Portal Search Results Link to Phenotypic Data, Images, & References
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(IMSR)
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