advanced cre-lox: generating reporters, inducible mice...

Advanced Cre-lox:

Generating Reporters,

Inducible Mice, and

Disease Models

Technical Information Services

December 8, 2016

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technical-support 4

How familiar are you with Cre-lox?

Moderately;

I have used very basic models and need some advice or

refresher training.

A little;

I have heard of it, but don’t know much about it.

I have used Cre-lox and want to learn more complex

modifications.

Learning Goals

Identify appropriate cre and floxed mouse

strains needed to generate reporter mice,

inducible mice and disease models

o Lox-STOP-Lox alleles

o Inducible Cre recombinases

Recognize whether a particular strain is

useful to generate the model

o Cre strain nomenclature

o Interpret the mouse description

Cre-lox Biology

oredP1 bacteriophage life cycle

Viral DNA is injected,

circularized using Cre-lox, and

replicated for development of

new viral particles

A Revolutionary Genetic Tool

Cre recombinase

catalyzes recombination

loxP site

34 bp DNA sequence

Location and orientation determines result:

o Deletion o Inversion o Translocation

Reviewed in: Nagy 2000 Genesis PMID:10686599

ATAACTTCGTATA-NNNTANNN-TATACGAAGTTAT

Abundant possibilities for genome manipulation!

Mechanism: Cre-lox Deletion

“floxed” target gene loxP loxP GeneX

Cre excision

knockout (null) allele

Tissue-specific Knockout Breeding

JAX® Mice |

Alb cre

GeneX loxP loxP

Cre-lox mouse:

Heterozygous for knockout (1st generation)

Alb cre

Liver-specific Cre B6.Cg-Tg(Alb-cre)21Mgn/J 003574

Homozygous floxed

GeneX loxP loxP

Alb cre

GeneX loxP loxP

Cre-lox mouse:

Heterozygous for knockout (1st generation)

Homozygous floxed

GeneX loxP loxP

25% homozygous for knockout (2nd generation)

Alb cre

GeneX loxP loxP

Alb cre

GeneX loxP loxP

Hemizygous Alb-cre Heterozygous floxed

Floxed Tg human SOD1G37R

Motor neuron- or

macrophage/microglia-

specific Cre

Develop

ALS-like pathology

Slower disease

progression

MN- or Mac/Micro-

KO SOD1G37R

Tissue-specific KO of Human Tg Amyotrophic Lateral Sclerosis

Boillée 2006 Science PMID: 16741123

B6.Cg-Tg(SOD1*G37R)1Dwc/J (016149)

Isl1-cre

(MN progenitors) Itgam-cre

(macrophage/microglia)

SOD1G37R expression in macrophages contributes to disease

Tissue-specific KO of human Tg Amyotrophic Lateral Sclerosis

Boillée 2006 Science PMID: 16741123

B6.Cg-Tg(SOD1*G37R)1Dwc/J (016149)

Learning Goals

Identify appropriate cre and floxed

mouse strains needed to generate

reporter mice, inducible mice and

disease models

Lox-STOP-Lox (LSL) Alleles

Basis for many cre disease models, cre-reporters,

optogenetics tools

Removal of Stop codon allows expression of

downstream gene

Promoter LoxP

Promoter

LoxP LoxP

X STOP GeneX

LSL-Reporter Breeding

x Nestin cre STOP LacZ loxP loxP

Reporter strain B6.129S4Gt(ROSA)26Sortm1Sor/J 003474

Nervous system-specific cre B6.Cg-Tg(Nes-cre)1kln/J 003771

LacZ loxP

JAX Cre Repository

Cre-Reporters

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

20X Lung

10X Heart

20X Skin

JAX Cre Repository Expression Data

Expression o E10.5, E15.5, P7 and P56 (adult)

o Whole-mount or sections

o Full necropsy (P7 & P56) – 11 organ systems, 30

organs/structures, 89 substructures

NanoZoomer slide scanner

Data shared with MGI Cre portal database

Predict the Phenotype of Offspring

loxP EGFP* tdTomato* STOP Gt(ROSA)26Sor loxP

CAG cre

A. ALL tissues will be both red and green

B. ALL tissues will turn red

C. SOME tissues will turn red

D. ALL tissues will turn green

E. SOME tissues will turn green

loxP EGFP*

loxP EGFP* tdTomato* STOP

Cre Reporter Variations Reporter Switching

B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J (007676)

Gt(ROSA)26Sor loxP

Gt(ROSA)26Sor

Cre Reporter Variations Reporter Switching

Muzumdar 2007 Genesis PMID: 17868096

Hepatocytes, days post-tamoxifen injection

STOCK Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J (007576), (B6 congenic version (007676))

crossed with CAG-Cre-Esr1, such as (004682) or (017595)

Cre Reporter Variations “Brainbow” Mice

Livet 2007 Nature PMID: 17972876

Tamoxifen-inducible

CAG-Cre transgenic

Cell autonomous

expression of RFP,

YFP & CFP

Neurons & some

astrocytes of the

dentate gyrus in the

hippocampus

Cre Reporter Variations “Brainbow” Mice

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

8 Tg copies & 90 colors B6;CBA-Tg(Thy1-Brainbow1.0)LLich/J (007910)

>8 Tg copies & 166 colors

3 Tg copies &10

colors

Cre Reporter Variations Brainbow Color Palette is Sensitive Tg copy number

Pdx1 cre

Pancreas-specific cre B6.FVB-Tg(Pdx1-cre)6Tuv/J 014647

Development of pancreatic tumors

LSL-Kras B6.129S4-Krastm4Tyj/J 008179

x STOP Kras G12D loxP loxP

Kras G12D loxP

Hingorani 2003 Cancer Cell PMID: 14706336

LSL-Oncogene Breeding

Ctrl Pdx1-Cre, KRAS G12D

Pancreatic Ductal Adenocarcinoma Tissue-Specific Oncogene Activation + Heterozygous KO

B6.FVB-Tg(Pdx1-cre)6Tuv/J (014647)

Hill 2010 Cancer Res. PMID: 20807812

Triple mutants develop acinar-ductal metaplasia

(ADM) that progresses to (PDAC)

Pdx1-cre

KrasG12D/+, Pten+/-

metaplasia

B6.129S4-Krastm4Tyj/J (008179)

C;129S4-Ptentm1Hwu/J (004597)

(B6 version: B6.129S4-Ptentm1Hwu/J 006440)

29 JAX Cre Repository |

Cre and Floxed Strains are Modular

Tissue Cre Driver Strain

Pancreas B6;CBA-Tg(Pda1-

cre)6Cvw/J 014647

Liver B6.Cg-Tg(Alb-

cre)21Mgn/J 003574

Prostate STOCK Tg(Pbsn-

cre)4Prb/J 026662

(melanocytes)

B6.Cg-Tg(Tyr-

cre/ERT2)13Bos/J 012328

Immune cells

(B cells)

B6.129P2(C)-

Cd19tm1(cre)Cgn/J 006785

Conditional

Allele

Strain

Oncogenic Kras

(KRASG12D)

B6.129S4-Krastm4Tyj/J 008179

Pten (tumor

suppressor)

B6.129S4-Ptentm1Hwu/J 006440

(tumor

suppressor)

STOCK Brca1tm1Aash/J 017835

Trp53 (tumor

suppressor)

B6.129P2-Trp53tm1Brn/J 008462

Oncogenic

BCR/ABL

C57BL/6-

Bcrtm1(BCR/ABL)Tsr/J 023520

600+ Cre-expressing strains and 800+ conditional alleles in our collection =

THOUSANDS of combinations

Learning Goals

Identify appropriate cre and floxed

mouse strains needed to generate

reporter mice, inducible mice and

disease models

Greco 2010 Development PMID: 20430743

Inducible Cre Fusion with Estrogen Receptor Ligand Binding Domain

*Control when Cre moves to the nucleus, when recombination occurs*

**Still dependent on when the promoter is active**

What is the final Cre genotype

needed to make an inducible KO?

Homozygous floxed

GeneX loxP loxP

Tamoxifen Inducible Cre

CAG Cre-ER

Hom Cre

Hemi Cre A

What is the final flox genotype

needed to make an inducible KO?

Homozygous floxed

GeneX loxP loxP

CAG Cre-ER

Hom floxed

Het floxed C

How many rounds of breeding to

make an inducible KO?

Homozygous floxed

GeneX loxP loxP

CAG Cre-ER

More than one

GeneX loxP loxP

Homozygous floxed Hemizygous Cre

Homozygous floxed

GeneX loxP loxP

2 Generations

Inducible Cre Breeding

Tamoxifen

Tamoxifen Inducible Cre B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J (004682)

CAG Cre-ER

4-OH-tamoxifen

Tamoxifen Dosing Options

IP injection

o Adults: 2-5 daily doses

o Pregnant females (embryos): 1-2 doses, co-

administered with progesterone to reduce abortion risk

Oral gavage

Drinking Water

o Often longer induction times (weeks vs. days)

o Weight loss common (10-15%)

Topical application (skin-specific cre mice)

tamoxifen

Establish the best dosage empirically!

Sample protocol

Pros and Cons

Induction Level May Vary with Dose

Hayashi 2002 Dev Biol PMID: 11944939

STOCK Tg(CAG-cre/Esr1*)5Amc/J (004453)

telencephalon

embryo

24 hr.

post-injection

Induction Level May Vary by Tissue

Hayashi 2002 Dev Biol PMID: 11944939

STOCK Tg(CAG-cre/Esr1*)5Amc/J (004453)

cortex

kidney

cerebellum

Adult mice injected 1/day for 5 days

Inducible Cre Considerations

Same considerations as “regular” Cre

o Tissue specificity, mosaicism

o Recombination efficiency of different floxed alleles

o Chromosomal location of Cre vs floxed alleles

o Cre toxicity

Tamoxifen dosing

o Route of administration, concentration, number of doses

o Timing before analysis, tissue turnover

Leakiness

Control selection

Availability of tools

Pdx1 cre

Pancreas-specific cre B6.FVB-Tg(Pdx1-cre)6Tuv/J 014647

Development of pancreatic tumors

LSL-Kras B6.129S4-Krastm4Tyj/J 008179

x STOP Kras G12D loxP loxP

Kras G12D loxP

Hingorani 2003 Cancer Cell PMID: 14706336

What tools are needed to make this inducible?

Ctrl Pdx1-Cre, KRAS G12D

Before 2015, this strain was not available.

024968

Doxycycline-inducible System

Promoter rtTA

tetO gene

Promoter tTA

tetO gene

NO Doxycycline

Tet On System (rtTA) Tet Off System (tTA)

Doxycycline-inducible System

Promoter rtTA

tetO gene

Promoter tTA

tetO gene

+ Doxycycline

Turns on gene expression Turns off gene expression

Tet On System (rtTA) Tet Off System (tTA)

Pdx1 cre

Doxycycline-inducible Cre Mice: Regulating rtTA and target expression with Cre

Gt(ROSA)26Sor loxP loxP STOP rtTA

B6.Cg-Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy/J 005670

Dow 2014 PLoS One PMID: 24743474

Belteki 2006 Nucleic Acids Res PMID: 15784609

Tet-ON KrasG12D

to model Pancreatic

Cancer tetO KrasG12D

Pdx1 cre

Cre + Gt(ROSA)26Sor loxP loxP STOP rtTA

X recombination

Gt(ROSA)26Sor loxP rtTA

tetO KrasG12D

Tet-ON KrasG12D

to model Pancreatic

Cancer

Pdx1 cre

recombination

Gt(ROSA)26Sor loxP loxP STOP rtTA

Gt(ROSA)26Sor loxP rtTA

tetO KrasG12D

Tet-ON KrasG12D

to model Pancreatic

Cancer

Learning Goals

Identify appropriate cre and floxed mouse

strains needed to generate reporter mice,

inducible mice and disease models

Recognize whether a particular strain

is useful to generate the model

Nomenclature

Tamoxifen inducible strains

o Promoter-Cre/Esr*1 B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J (004682)

o Promoter-Cre/ERT B6;129-Gt(ROSA)26Sortm1(cre-ERT)Nat/J (004847)

o Promoter-Cre/ERT2 Higher sensitivity to tamoxifen (~ 10-fold)

B6.129-Gt(ROSA)26Sortm1(cre-ERT2)Tyj/J (008463)

Doxycycline inducible

o Promoter-rtTA Tet-ON

B6.Cg-Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy/J (005670)

o Promoter-tTA Tet-OFF

B6.129P2(Cg)-Gt(ROSA)26Sortm1(tTA)Roos/J (011008)

https://www.jax.org/mouse-search

Featured: Cre expressing, Cre-reporter

Products: Live mice, Frozen Embryos

Stock type: Transgenic, Targeted

Mutation

Attribute: Null/KO, Reporter, Inducible,

Conditional ready/floxed

Searching for Cre-lox Strains Using filters…

Searching for Cre-lox Strains …and Keywords

Searches text found in the datasheets

o Strain name, gene and allele information,

strain description and development, site of

expression, disease & phenotype terms,

research area and donating investigator.

o “widespread,” “green,” “LSL”

Use the autocomplete terms whenever

possible (gene names, disease terms)

Try synonyms

https://www.jax.org/mouse-search

Searching for Cre-lox Strains

Strain Datasheet

What the mouse is, overall purpose (original intent)

How it was made (molecular biology, strains used to

create)

Controls (genetic background)

References

o Selected = original research paper and others that

characterize the model

o Additional = others who have used and cited this strain,

or strains similar to it

Strain Datasheet

CRISPR/Cas9 model generation

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Mouse genome scanning

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Basic and complex mouse breeding,

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Humanized mice, patient-derived

xenograft preclinical models and

therapeutic drug evaluation

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