Genetic Engineering Unlocked: Cre-lox & Beyond

Genetic Engineering

Unlocked:

Cre-lox & Beyond

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Presented by:

Brian W. Soper, PhD

Technical Information Services

Hosted by Charles River, exclusive

distributor of JAX® Mice in 27 European

Countries

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

ATAACTTCGTATAGCATACATTATACGAAGTTAT

Abundant possibilities for genome manipulation!

Cre

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http://www.ncbi.nlm.nih.gov/pubmed?term=10686599

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

Alb cre

GeneX

GeneX

LoxP GeneX LoxP

GeneX LoxP LoxP

Liver-specific cre transgene Ex: B6.Cg-Tg(Alb-Cre)21Mgn/J

(003574)

Homozygous “floxed” mouse

x

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http://jaxmice.jax.org/strain/003574.html

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


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Cre-Lox Tissue-Specific Knockout (continued)

Hemizygous Alb-cre heterozygous “floxed” gene

Alb cre

GeneX

LoxP GeneX LoxP

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LoxP GeneX LoxP

GeneX LoxP LoxP

Alb cre

GeneX

LoxP GeneX LoxP x


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25% homozygous for gene X conditional knockout (2nd generation)

LoxP GeneX LoxP

GeneX LoxP LoxP

GeneX

Alb cre

GeneX

LoxP LoxP

LoxP LoxP

Alb cre

GeneX

LoxP GeneX LoxP x


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LoxP GeneX LoxP

GeneX LoxP LoxP

GeneX

Alb cre

GeneX

LoxP LoxP

LoxP LoxP

Alb cre

GeneX

LoxP GeneX LoxP


25% homozygous for gene X conditional knockout (2nd generation)

x


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Improving Conditional Knockout Efficiency

x

12.5% conditional gene X knockouts

Heterozygous null mouse

(traditional knockout)

GeneX LoxP LoxP

Alb cre

GeneX

LoxP GeneX LoxP

LoxP GeneX LoxP

Alb cre

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Improving Conditional Knockout Efficiency

LoxP GeneX LoxP

Alb cre

Hemizygous Alb-cre heterozygous null mouse

25% conditional gene X knockouts

x GeneX LoxP LoxP

LoxP GeneX LoxP


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Alb cre

GeneX

x Homozygous

“floxed” mouse

GeneX

LoxP GeneX LoxP

oocyte-specific cre expression

Ex: C57BL/6-Tg(Zp3-cre)93Knw/J

(003651)

ZP3 cre

Ovary

ZP3 cre

Oocytes

Cre-Lox Germline Knockouts

2 more generations to produce

homozygote null mouse 18


Homozygous LoxP mouse Cre mouse – cre transgene (Tg)

early, widespread expression promoter

B6.Cg-(Sox2-cre)1Amc/J

(008454)

Offspring: 50% heterozygous knockout after 1 generation

LoxP GeneX LoxP

LoxP GeneX LoxP

GeneX

Cre

GeneX

GeneX

Sox2

Cre-Lox Knockout Breeding Scheme

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

LoxP GeneX LoxP

LoxP GeneX LoxP

Alb Cre

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Inducible Cre Mouse Models

x Homozygous LoxP mouse Inducible Cre mouse –

tamoxifen dependent Cre function

Ex: B6.Cg-Tg(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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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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http://www.ncbi.nlm.nih.gov/pubmed/23169059


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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http://www.ncbi.nlm.nih.gov/pubmed/23169059


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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JAX Cre Reporter Strains

Livet J et al. 2007. Nature 450(7166):56-62. PMID:17972876

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Cre Reporter Strains

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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http://www.jax.org/search/Main.jsp?qt=003474&x=18&y=13

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: heart-specific cre transgenic

**Could be any Cre recombinase strain

Cre

LacZ stain confirms Cre activity in expected tissues

LoxP LoxP LacZ

LoxP LoxP LacZ

Myh6

1 Generation

LoxP LacZ

Cre Myh6

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Other Cre Reporter Variations Reporter switching

Promotor LoxP LoxP GFP LacZ

Promotor LoxP GFP

STOP

Ex. STOCK Tg(ACTB-Bgeo/GFP)21Lbe/J (003920)

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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

LoxP STOP Kras G12D

e.g. STOCK Tg(MMTV-cre)4Mam/J

(003553)

LoxP

LoxP Kras G12D

Mice develop

mammary neoplasms

B6.129S4-Krastm4Tyj/J (008179)

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Other Cre-Lox Variations Conditional Transgene with Reporter

Promotor LoxP LoxP TransgeneX LacZ

Promotor LoxP TransgeneX

STOP

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Other Cre-Lox Variations Conditional Transgene with Fusion Protein

Reporter

Promotor LoxP LoxP TransgeneX

Promotor LoxP

GFP

TransgeneX GFP

STOP

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Other Cre-Lox Variations Conditional transgene with reporter

Promotor LoxP LoxP

Promotor LoxP

STOP TransgeneX GFP IRES

TransgeneX GFP IRES

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Other Cre reporter Variations: “Brainbow” Mice

Multiple fluorescent protein

sequences

Pairs of incompatible

LoxP sites

LoxP sites alternated to

create mutually-exclusive

recombination events

Following cre excision, one

of 3 outcomes (colors)

possible in cre expressing

cells/tissues


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“Brainbow” Mice

Tamoxifen-inducible

CAG-Cre transgenic

Cell autonomous

expression of RFP,

YFP & CFP

Neurons & some

astrocytes of the

dentate gyrus in the

hippocampus


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“Brainbow” Mice


Multiple copies & multiple integrations of

the Brainbow transgene

B6.Cg-Tg(Thy1-Brainbow1.0)HLich/J (007901)

8 transgene copies & 90 color variations

B6;CBA-Tg(Thy1-Brainbow1.0)LLich/J

(007910) >8 transgene copies & 166 color

variations

Like pixels on a TV screen—combinations of fluorophores produce expanded color palettes

3 transgene copies &

10 color variations

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Improved Fluorescent Cre Reporters

Ai3 B6.Cg-Gt(ROSA)26Sortm3(CAG-EYFP)Hze/J (007903)

Ai2 B6.Cg-Gt(ROSA)26Sortm2(CAG-EYFP)Hze/J (007920)

Ai9 B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (007905)

Ai9 B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (007909)

Ai14 B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (007914)

Ai6 B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J (007906)

Madisen L et al. 2010 Nat Neurosci 13(1): 133-40. PMID:20023653

Dr. Hongkui Zeng, Allen Institute for Brain Science

http://transgenicmouse.alleninstitute.org/

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Allen Institute for Brain Science New cre reporters x brain-specific cre transgenics


B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (007909) x

B6.Cg-Tg(Camk2a-cre)T29-1Stl/J (005359)

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Allen Institute for Brain Science


B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J (007906) x

FVB-Tg(GFAP-cre)25Mes/J (004600)

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JAX Cre Repository

Largest collection of Cre-Lox strains

330+ cre-expressing

130+ inducible Cre strains

480+ floxed genes

90+ floxed Stop Cre reporters

Importing over 200 new neuronal specific Cre strains

http://cre.jax.org/NeuroCres.html

http://cre.jax.org

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http://cre.jax.org/

http://cre.jax.org/index.html

Locating JAX Cre Strains

www.jaxmice.jax.org/query

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http://jaxmice.jax.org/query

http://jaxmice.jax.org/query

JAX Cre Repository http://cre.jax.org

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http://cre.jax.org/

Search by keyword within browser; type “ctrl + F”

JAX Cre Repository Locating Strains by Tissue Expression Site/Promoter

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JAX Cre Repository http://cre.jax.org

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http://cre.jax.org/

Cre Expression Data

http://cre.jax.org/data

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Cre Expression Data


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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 50


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 Creportal 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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http://www.creportal.org/

Cre Portal Search Results Link to Phenotypic Data, Images, & References

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Tissue Specificity of Recombinase

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Genetic Engineering Unlocked: Cre-lox & Beyond

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