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EUCOMM: Tools for Functional Annotation of the Mouse Genome
Cre-driversEUCOMMTOOLS
European Conditional Mouse Mutagenesis Program:
TOOLS for Functional Annotation of the Mouse Genome
IMPC meeting28th-29th September, 2011
Washington, USA
Wolfgang Wurst
Helmholtz Zentrum Munich
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
The Challenge •20,000 floxed alleles•Integrated database
•Hundreds of Cre driverstrains
••VS
acriat
otuered
s badcaktgaroubas
ned s
•Various background
IMPC Meeting, 28th-29th September, Washington, USA
cover all or ns and cell t s
EUCOMM: Tools for Functional Annotation of the Mouse Genome
Objectives:
» Identification of 500 genes and their promoters suitable as Cre drivers covering all organs and major cell types ing ga major ype
» Establishment of 500 BAC-CreERT2T2A-eGFP/BAC-Cre-T2A-eGFP
(in the Rosa26 or Hprt locus) constructs and recombinant ES cells Cre
driver transgenic mouse lines
» Establishment of 250 BAC-CreERT2T2A-eGFP/BAC-Cre-T2A-eGFP
driver transgenic mouse lines
» Archiving and distribution by EuMMCR/EMMA
IMPC Meeting, 28th-29th September, Washington, USA
AAliliccaannttee
Helmholtz Zentrum München W. Wurst (coordinator), A. Hörlein, Hrabe de Angelis
nez
National Research
MRC/HGU R. Baldock
Wellcome Trust Sanger Institute A. Bradley (coordinator), W. Skarnes, R. Ramirez-Solis EMBL/EBI
P. Flicek, D. Smedley
University of Dresden F. Stewart EdEdininbbuurgrg
MRC S. Brown
Institute Clinique de la Souris (ICS) Y. Herault
UMH S. Marti
CouncilG. Tocchini-Valentini
EMBL N. Rosenthal
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
Cre
Dri
ver
RC
reD
river
R
WP5:
ES
Cell
Reso
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ell
Reso
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EUCOMM: Tools forEEUCUCOOMMTMMTOOOOLLSSFunctional Annotation
SSTTRURUCTCTURUREE C
re D
river
Reso
urc
es
of the Mouse Genome
WP1: Gene selection
WP2: Vector design and construction
WP3: Technology
WP4: development
ES cell production
WP5: Generation of transgenic mouse lines
WP6: Cre Expression Annotation
WP7: Phenotyping/Cre transgenic mice
WP8: Bioinformatics
WP9: International Coordination & Databases
WP1: Gene selection
WP2: Vector design and construction
WP4: ES cell productionES cell production
WP5: Generation of transgenic mouse lines
WP10: Distribution & Archiving
Co
nd
ition
al E
S C
ell R
eso
urc
e
Management; WP 11: ‘Training, Sustainability, Dissemination and Outreach’ IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
WP1 Gene/Promoter Selection:
(Damian Smedley, Nadia Rosenthal)
» Create – categorize existing Cre lines
Create survey - collecting requests from the scientific community Create survey - collecting requests from the scientific community
Coordinate production with e.g. NorCOMM2, CanEuCre, Pleiades,NIH Blueprint, Allen Brain Institute, Riken, Jax
Experts opinions and suggestions
Identify specific expression - Eurexpress (embryo), GXD (adult), Sanger LacZ
Remake most popular cre lines not on C57BL6/N avaibale
»»
»
»
»
»
» More than 250 genes have been selected
IMPC Meeting, 28th-29th September, Washington, USA
Overall summary of Cre driver selection
ANALYSIS DRIVERS
Suggested genes from survey 17
Suggested tissues from survey 18
SpecifSpe icif c aduic ad lu t expression lt expression identiden it fi if ed from Eurexpress ied from Eurexpress 64 64
Specific adult expression identified from GXD 94
Specific adult expression identified from BioGPS 71
Sanger LacZ expression 11
Popular Cre lines to remake as inducible B6N 41
Tissues not covered by current Cre lines 7
Expert suggestions 26
TOTAL 349
EUCOMM: Tools for Functional Annotation of the Mouse Genome
IMPC Meeting, 28th-29th September, Washington, USA
» u e t ose w t a re ne n t e , or pro ects
EUCOMM: Tools for Functional Annotation of the Mouse Genome
Selection status: » Curated to a non-redundant list of 275 genes with official MGI
symbols and IDs
» Excluded those with a known Cre line at www.creline.org
» Excl d d h i h C li i h GENSAT CDN ICS jExcluded those with a Cre line in the GENSAT, CDN or ICS projects
» Identify specific expression in normal adult samples at GXD, ArrayExpress and BioGPS (GNF microarray datasets) with possible backup from Eurexpress embryonic expression patterns
IMPC Meeting, 28th-29th September, Washington, USA
http://www.creline.org/
(1)
EUCOMM: Tools for Functional Annotation of the Mouse Genome
Strategy: Cre Knock-ins
» Exploit EUCOMM/KOMP Modular Vector Resources to rapidly build a set of Cre Knock-ins
» Mixture of re-targeting and RMCE » Mixture of re-targeting and RMCE
» Using Cre and inducible CreERT2
» CreERT2 Knock-in vector design
» Vector includes nlsEGFP reporter
» T2A or LF2A for signal sequence containing genes
» Comparison of Targeted Knock-ins versus BAC RMCE
» Coordination with Haydn Prosser from Bradley laboratory IMPC Meeting, 28th-29th September, Washington, USA
``
EUCOMM: Tools for Functional Annotation of the Mouse Genome
» CreERT2 Gateway/RMCE Cassette:
attL1 FR LF2A LF2A Rox Rox loxP attL2 T
SA nlsEGFP CreERT2 pA PGK:pur pA `
GATEWAY
RMCE
Barry Rosen, Bill Skarnes
IMPC Meeting, 28th-29th September, Washington, USA
cess
(1)
EUCOMM: Tools for Functional Annotation of the Mouse Genome
Considerations of EUCOMMTools Cre Knock-ins
» Simple to make with existing technology and resources
» Pro’s of Cre-Knock-ins •• R Ree--uuttiilliizazattiioon n oof f pprreevivi oouuslsl y y vavalliidd aatteedd mater a s, t us g suc
rate – No new technology needed, only CreERT2 vector cassette
• Knocked-in Cre should faithfully recapitulate endogenousexpression
» Con’s of Cre Knock-ins • Difficult to breed animals to homozygosity in most cases
• Potential for haplo-insufficient phenotypesIMPC Meeting, 28th-29th September, Washington, USA
1 x xA ACreIRES Bact::neo
attL Ro Ro attL2 1 IRES Cre x pA p Bact::neo pA p x
attL1 LF2A Ro Ro attL2 Cre x pA Bact::neo pA x
EUCOMM: Tools for Functional Annotation of the Mouse Genome
Alternative Cre Alleles
3’UTR IRES Cre Insertion Leaves endogenous protein intact Leaves endogenous protein intact
2A Mediated Cre 3’ Insertion N-terminal fusion, adds COOH extension to endogenous protein
IMPC Meeting, 28th-29th September, Washington, USA
loxP loxP
5’ 1 2 UTR
T2AT2A loxP loxP
1 Bact::neo 25’ Cre UTR
T2A loxP
1 2 5’ Cre UTR
T2A
-COOH NH2-Pro w.t. protein -COOH NH2- Cre
(1)
EUCOMM: Tools for Functional Annotation of the Mouse Genome
Alternative Cre alleles: 5’ Cre 2A-mediated insertion
Target Locus
Targeted 5’ Cre knock-in
Cre deletion of marker
Protein translation
IMPC Meeting, 28th-29th September, Washington, USA
,’
(1)
EUCOMM: Tools for Functional Annotation of the Mouse Genome
Pros and Cons of Different Alternative Cre Alleles ConPro
-Applicable to all genes Universal LF2ACre Knock- -Still deletes an allele -Exploits existing IKMC resources
in -Applicable to all genes -Reduced Cre from IRES?
33 UTR IRES Cre Insertion ’UTR IRES Cre Insertion --Endogenous protein intact Endogenous protein intact -new vector no RMCE -new vector,no RMCE -Validated in literature -HT vector construction
-Adds 3’ aa’s to
2A Mediated Cre 3’ Insertion-Should express high levels endogenous protein of Cre -Additional aa’s with LF2A
-new vector, no RMCE
-Initiating ATG must -Minimal perturbation of 5’ Cre 2A-mediated be defined carefully locus
-new vector, no insertion -Promoter specific RMCE
applications IMPC Meeting, 28th-29th September, Washington, USA
(1)
EUCOMM: Tools for Functional Annotation of the Mouse Genome
CreERT2 Knock-ins: Progress Re-Targeting
» 49 Intermediate Vectors Identified
» Criteria: Targeted ES Cells Status in EUCOMM/KOMP
»» Avoid signal sequence/secreted for moment(50% of genes) Avoid signal sequence/secreted for moment(50% of genes)
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
EUCOMMTOOLS – BAC knock-in Technology (Hayden Prosser, Allan Bradley)
RMCE strategy for introducing BAC eGFPT2ACreERT2/Cre-T2A-eGFP transgenes into the ES cell genome
IMPC Meeting, 28th-29th September, Washington, USA
- “ ”
EUCOMM: Tools for Functional Annotation of the Mouse Genome
Cre-BAC transgenesis by site specific integration
Pros: » Can use Human or Mouse BACs » Avoids disruption of endogenous alleles as would occur in targeted
Cre knockknock-in to “Driver” gene river gene Cre in to D
» Avoids variation that might occur with random integration » Cassette exchange removes extraneous prokaryotic vector DNA
Cons: » Integrated BACs do exhibit deletions in a proportion of cases » “Passenger” genes may be associated with Cre driver transgene » Gene size limits
IMPC Meeting, 28th-29th September, Washington, USA Joint EUCOMM-CREATE-EUCOMMTOOLS Progress Meeting, July 5-7, 2011, Rome
EUCOMM: Tools for Functional Annotation of the Mouse Genome
» Currently:
» Direct comparison of Knock-in versus Bac-knock-in strategy!
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation f the Mouse Genome
T2
WP6: Cre Expression oAnnotation: Salvador Martinez
Prom-eGFP-CreER X Rosa26 reporter
• Immunohistochemistry on eGFP • Saggital, longitudinal, transverse tissue
section • anti-GFP antibody, lacZ
immunohistochemistry, cell type specific markers
• Annotation – Edinburgh Mouse Atlas (EMAP), Allen Brain Atlas, Eurexpress
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
Distribution of EUCOMMTOOLS Material
Dissemination of • mutant ES cell lines http//:www.eummcr.org
• mouse lines http//:www.emma.org
Dissemination asap as QC confirmed
IMPC Meeting, 28th-29th September, Washington, USA
http:http//:www.emma.orghttp:http//:www.eummcr.org
EUCOMMTOOLS integration in IKMC portal (www.knockoutmouse.org)
EUCOMM: Tools for Functional Annotation of the Mouse Genome
Long term SUSTAINABILITY
IMPC Meeting, 28th-29th September, Washington, USA
http:www.knockoutmouse.org
EUCOMM: Tools for Functional Annotation of the Mouse Genome
» Outlook:
» Technology and reagent development » IKMC toolkit extension
» New ligand/SSR-LBD pairs
» New conditional switches
» Additional optogenetic tools, markers, cell survival
IMPC Meeting, 28th-29th September, Washington, USA
MRC/HGU R. Baldock
Wellcome Trust Sanger Institute A. Bradley (coordinator), W. Skarnes, R. Ramirez-Solis EMBL/EBI
P. Flicek, D. Smedley
University of Dresden F. Stewart EdEdininbbuurgrg
MRC S. Brown Helmholtz Zentrum München
W. Wurst (coordinator), A. Hörlein, Hrabe de Angelis
Institute Clinique de la Souris (ICS) Y. Herault
UMH S. Martinez AAliliccaannttee
National Research CouncilG. Tocchini-Valentini
EMBL N. Rosenthal
IMPC Meeting, 28th-29th September, Washington, USA
EUCOMM: Tools for Functional Annotation of the Mouse Genome
EUCOMM: Tools for Functional Annotation of the Mouse Genome
IMPC Meeting, 28th-29th September, Washington, USA
Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Strategy: Cre Knock-insSlide Number 10Considerations of EUCOMMTools Cre Knock-insAlternative Cre AllelesAlternative Cre alleles: 5’ Cre 2A-mediated insertionPros and Cons of Different Alternative Cre AllelesCreERT2 Knock-ins: Progress Re-TargetingSlide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32