cre-lox basics: generating knockout mice webinar...
TRANSCRIPT
Cre-lox Basics:
Generating
Knockout Mice
Technical Information Services
March 21, 2017
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Learning Goals
Identify components of the Cre-lox system and understand the mechanism of recombination
Design a typical breeding strategy to generate tissue-specific and germline knockout mice using Cre-lox
Identify potential pitfalls with Cre-lox
Search for strains and find cre expression characterization data
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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
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
Mechanism: Cre-lox Translocation
Cre
Chr 3
Chr 6 LoxP
LoxP
Reciprocal
Translocation (3;6) LoxP
LoxP
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Tissue-Specific Knockout Breeding
JAX® Mice |
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Alb cre
GeneX loxP loxP
GeneX
Cre-lox mouse:
Heterozygous for knockout (1st generation)
Alb cre
GeneX
GeneX
Liver-specific Cre B6.Cg-Tg(Alb-cre)21Mgn/J 003574
x
Homozygous floxed
GeneX loxP loxP
GeneX loxP loxP
Alb cre
GeneX loxP loxP
GeneX
Tissue-Specific Knockout Breeding
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Cre-lox mouse:
Heterozygous for knockout (1st generation)
x
Homozygous floxed
GeneX loxP loxP
GeneX loxP loxP
Tissue-Specific Knockout Breeding
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25% homozygous for knockout (2nd generation)
Alb cre
GeneX loxP loxP
GeneX loxP loxP
Alb cre
GeneX loxP loxP
GeneX
Hemizygous Alb-cre Heterozygous floxed
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Cre-lox Tissue-Specific Knockout (continued)
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Alb-cre +
loxP + loxP +
Hemizygous
cre
Alb cre
GeneX loxP loxP
GeneX
Homozygous floxed
GeneX loxP loxP
GeneX loxP 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
25% homozygous for gene X conditional knockout (2nd generation)
Hemizygous
cre
Alb cre
GeneX loxP loxP
GeneX
Homozygous floxed
GeneX loxP loxP
GeneX loxP loxP
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: Up to 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 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 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)
Promoter LoxP LoxP lacZ
X
Promoter LoxP lacZ
STOP
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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 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 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
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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Genotyping Strategies for loxP
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“floxed” target gene loxP loxP GeneX
X G
en
eX
Cre excision
Cre
knockout (null) allele
loxP
GeneX
loxP
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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
JAX Cre Repository*
Largest collection of Cre-lox strains
650+ cre-expressing
270+ inducible Cre strains
1100+ 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 October 2016
Searching for Cre Strains
https://www.jax.org/mouse-search 28
muscle
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Searching for Cre Strains
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Finding Cre-reporter strains
JAX® Mice |
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www.jax.org/mouse-search
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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
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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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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Learning Goals
Identify components of the Cre-lox system and understand the mechanism of recombination
Design a typical breeding strategy to generate tissue-specific and germline knockout mice using Cre-lox
Identify potential pitfalls with Cre-lox
Search for strains and find cre expression characterization data
37
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