advanced cre-lox: generating reporters, inducible mice...
TRANSCRIPT
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?
5
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.
Very;
I have used Cre-lox and want to learn more complex
modifications.
A
B
C
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
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Cre-lox Biology
oredP1 bacteriophage life cycle
Viral DNA is injected,
circularized using Cre-lox, and
replicated for development of
new viral particles
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cre
image
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!
Cre
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Mechanism: Cre-lox Deletion
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“floxed” target gene loxP loxP GeneX
X G
en
eX
Cre excision
Cre
knockout (null) allele
loxP
GeneX
loxP
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
Floxed Tg human SOD1G37R
Motor neuron- or
macrophage/microglia-
specific Cre
Develop
ALS-like pathology
Slower disease
progression
x
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)
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Isl1-cre
(MN progenitors) Itgam-cre
(macrophage/microglia)
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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
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
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Lox-STOP-Lox (LSL) Alleles
Basis for many cre disease models, cre-reporters,
optogenetics tools
Removal of Stop codon allows expression of
downstream gene
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Promoter LoxP
Promoter
LoxP LoxP
X STOP GeneX
GeneX
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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
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JAX Cre Repository
https://www.jax.org/research-and-faculty/tools/cre-repository
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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
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Predict the Phenotype of Offspring
loxP EGFP* tdTomato* STOP Gt(ROSA)26Sor loxP
x
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
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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
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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)
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Cre Reporter Variations “Brainbow” Mice
Livet 2007 Nature PMID: 17972876
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Tamoxifen-inducible
CAG-Cre transgenic
Cell autonomous
expression of RFP,
YFP & CFP
Neurons & some
astrocytes of the
dentate gyrus in the
hippocampus
Livet 2007 Nature PMID: 17972876
Cre Reporter Variations “Brainbow” Mice
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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
Livet 2007 Nature PMID: 17972876
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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
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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+/-
Human
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
Skin
(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
Brca1
(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
x
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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
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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**
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What is the final Cre genotype
needed to make an inducible KO?
x
Homozygous floxed
GeneX loxP loxP
GeneX loxP loxP
Tamoxifen Inducible Cre
CAG Cre-ER
Hom Cre
Hemi Cre A
B
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What is the final flox genotype
needed to make an inducible KO?
x
Homozygous floxed
GeneX loxP loxP
GeneX loxP loxP
Tamoxifen Inducible Cre
CAG Cre-ER
Hom floxed
Het floxed C
D
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How many rounds of breeding to
make an inducible KO?
x
Homozygous floxed
GeneX loxP loxP
GeneX loxP loxP
Tamoxifen Inducible Cre
CAG Cre-ER
More than one
One E
F
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GeneX loxP loxP
GeneX loxP loxP
Homozygous floxed Hemizygous Cre
x
Homozygous floxed
GeneX loxP loxP
GeneX loxP loxP
2 Generations
Inducible Cre Breeding
Tamoxifen
Tamoxifen Inducible Cre B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J (004682)
CAG Cre-ER
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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
Chow
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
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Induction Level May Vary with Dose
Hayashi 2002 Dev Biol PMID: 11944939
STOCK Tg(CAG-cre/Esr1*)5Amc/J (004453)
heart
telencephalon
gut
embryo
24 hr.
post-injection
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Induction Level May Vary by Tissue
Hayashi 2002 Dev Biol PMID: 11944939
STOCK Tg(CAG-cre/Esr1*)5Amc/J (004453)
cortex
heart
kidney
lung
liver
cerebellum
Adult mice injected 1/day for 5 days
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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
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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
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Doxycycline-inducible System
Promoter rtTA
tetO gene
Promoter tTA
tetO gene
NO Doxycycline
Tet On System (rtTA) Tet Off System (tTA)
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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)
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Pdx1 cre
1
Doxycycline-inducible Cre Mice: Regulating rtTA and target expression with Cre
2
Gt(ROSA)26Sor loxP loxP STOP rtTA
X
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
3
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Pdx1 cre
Cre + Gt(ROSA)26Sor loxP loxP STOP rtTA
X recombination
Gt(ROSA)26Sor loxP rtTA
tetO KrasG12D
Doxycycline-inducible Cre Mice: Regulating rtTA and target expression with Cre
Tet-ON KrasG12D
to model Pancreatic
Cancer
B6.Cg-Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy/J 005670
Dow 2014 PLoS One PMID: 24743474
Belteki 2006 Nucleic Acids Res PMID: 15784609
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+ Dox
Pdx1 cre
Cre +
recombination
Gt(ROSA)26Sor loxP loxP STOP rtTA
X
Gt(ROSA)26Sor loxP rtTA
tetO KrasG12D
Doxycycline-inducible Cre Mice: Regulating rtTA and target expression with Cre
Tet-ON KrasG12D
to model Pancreatic
Cancer
B6.Cg-Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy/J 005670
Dow 2014 PLoS One PMID: 24743474
Belteki 2006 Nucleic Acids Res PMID: 15784609
47
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
48
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)
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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…
50
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
51
Searching for Cre-lox Strains
52
Strain Datasheet
53
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
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Strain Datasheet
55
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