Cre-lox Basics: Generating Knockout Mice Webinar 21MAR2017jackson.jax.org/rs/444-BUH-304/images/Cre-lox-Basics-Webinar-21MAR2017.pdf · Tissue-Specific Knockout Breeding JAX® Mice

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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