Institut Clinique de la Souris
Yann Herault CNRS, INSERM, Université de Strasbourg,
1 rue Laurent Fries - 67404 Illkirch, France. [email protected]
The mouse clinics, improving phenotyping
Y. Herault, Veyrier du Lac 11-10-11
Mouse Housing
and Distribution
Abdel AYADI
Clinical
Phenotyping
Tania SORG
Mutagenesis and
Transgenesis
Guillaume PAVLOVIC Marie-Christine BIRLING
Department for Mutagenesis and Transgenesis
A large panel of models available
Various ES Cell lines:
Validated, highly germline competent (C57BL/6N, 129S2, BALB/c) Molecular Services
• Validation of genetic
modifications
• RNAi knockdown
profiling
Mouse models “A la carte”
Define strategy and feasibility Vector construction
LoxP>
LoxP>
1.6 kb3.5 kb
ex2
ex3
ex4
ex5
ex6ex7
ex8FRT>
NEO
FRT>
ex9
ex10
ES cell culture Microinjection to GLT
3
Delivery of 1006 personalized projects between 2002-10 New resources for the scientific community
The CreERT2 Zoo (50 lines in dev.) Conditional allele for all 49 mouse nuclear receptors and many of their cofactors: 43 « floxed » NR alleles already made
RARa, RARb, RARg, PPARa, PPARb, PPARg, RevErba, RevErbb, RORa, RORb, RORg, VDR, HNF4a, HNF4g, RXRa, RXRb, RXRg, TR4, ERa, ERb, ERRa, ERRb, ERRg, AR, PR, GR, MR, NUR77, NURR1, LRH1, SF-1, SHP, CAR, PNR, GCNF1, PXR, LXRa, LXRb, FXRa, FXRb,
EAR2, TLX, COUP-TF2
6 « floxed » NR alleles in progress TR2, DAX1, COUP-TF1, NOR1, TRa, TRb
9 « floxed » co-factor alleles available REA, Trip-15, Rap-250, Trap-220, NcoR1, NcoR2, SRCx, TIF2
2 « floxed » co-factor alleles in progress PGC-1a, PGC-1b
http://www.ics-mci.fr/crezoo.html http://www.ics-mci.fr/nrzoo.html
Department for Mutagenesis and Transgenesis
4
Autoclaves
Lab
A. Ayadi
Phenotyping area (900m2) 8 animal rooms dedicated for mice housing (1 500 cages) Laboratories for mice phenotyping Autoclave
Breeding area (2100 m2) 12 animal rooms (16 640 cages) Microinjection Unit and dedicated nursery rooms (184 m2) Cryopreservation Unit Washing area (285m2) (washing tunel, rack washer, autoclaves) Storage area (115m2)
Department for Mouse Housing and Distribution
5
Metabolism 22%
Cardio-resp 6%
Behavior and Cognition
19%
Anapath 24%
Biochemistry and
hematology 29%
2009
Services Capacity/year Metabolism 200
Biochemistry and Hematology
200
Anatomo-Histopathology 200
Cardio-respiration 200
Behavior, cognition, sensory organs
150
>250 Standardized tests in 5 Services
Department for Phenotyping Head T. Sorg
6
Examples of Specialised phenotyping pipeline: Metabolism, Cardiology, Histology
8
5 weeks-old 6 7 8 9 10 -------- 16 17 18 19 20 21 22 23
qNMR
Dysmorphological screen
OGTT
Body Lean and Fat Content Blood collection Food consumption and Body weight Glucose Tolerance & Insulin Sensitivity test Cardiology Bone Histopathology
IPIST (optional)
Non-invasive blood pressure
Body weight every week until week 16
Blood collection (glucose, adipokines, lipids)
Echocardiography
Mice fed with Chow diet Metabolic Testing, Aging, Mice fed with HF-HS diet
qNMR
Dexascan
Sacrifice :
Blood collection (adipokines, lipids, plasma enzymatic activities, plasma chemistries, hematology)
Tail biopsy
Necropsy exam
Histopathology (targeted analysis)
Arrival & Acclimation
Functions / Parameters
8
Examples of specialized phenotyping pipeline: Behavior & Cognition
7 weeks-old 10 11 12 13
Open field Test
Acoustic startle reflex and PPI
Tail suspension
Shock threshold
Tail Flick and Hot Plate Test
PTZ susceptibility
Test order: from least to most stressful
Fear conditioning
Y-maze
Clinical observations
Grip Test
String Test
Rotarod Tail biopsy
Arrival & Acclimation
Anxiety Sensory motor functions Learning and memory Depression Schizophrenia Pain sensitivity Epilepsy
Functions
9
• Gene function and target validation
• Model characterization
• Possibility to combine phenotyping to pharmacological treatment, in wildtype or mutant models
• Lead selection
• New indication or combination indication
• Drug genetic projects
Mutagenesis, Phenotyping & Pharmacological studies
10
> 250 tests fully validated & standardized suitable for HTP first line analysis or more in depth secondary analysis with expert consulting, data analysis & report
The French National Infrastructure for Mouse PHENOGENOMICS
CIPHE
TAAM
ICS
M. Selloum, M.-C. Birling G. Pavlovic, A. Ayadi, T. Sorg, Y. Herault
C. Olivier, S. Lerondel P. Lopes, C. Fremond,
F. Fiore, B. Malissen,
WP2
Mutagenesis WP3
Zootechny WP4
Phenotyping
WP1: Management
WP5: Discover of mammalian genes function /
Contribution to IMPC
USE
RS/
PAR
TNER
S
USE
RS/
PAR
TNER
S
Scientific Experts: Block-Zupan A., Chambon P., Chan S., Fiore F., Frossard N., Gorvel J.-P., Grenot P., Hérault Y., Kieffer B., Le Pape A., Laporte J., Malissen B., Malissen M., Mark M., Metzger D., Monassier L., Ouagazzal A.M., Pourquié O., Ricci R., Romand R., Roux M., Spitz F., Ugolini S., Vivier E., …
The way forward the Functional Annotation of the Mammalian Genome
The International Mouse Phenotyping Consortium
Chair: S. Brown
http://www.mousephenotype.org/
Launch Sept 28th 2011
www.mousephenotype.org
Global Phenotyping - The Context
The function of the majority of the genes in the mouse (and human) genomes is unknown
We are remarkably poor at predicting the functions of genes – pleiotropy will be key to understanding systems
KOs have been generated and analysed in only some 30% of mouse genes
Data for these genes is patchy – and is dependent on the experience and interests of the investigator
www.mousephenotype.org
promoter
start codon
stop codon
prom-Neo
FRT loxP FRT loxP loxP SA-lacZ-pA
promoter loxP FRT SA-lacZ-pA
lacZ
Critical exon
tm1a
tm1b (post-Cre)
KO first allele (reporter-tagged insertion allele) Cre
promoter loxP FRT loxP
tm1c (post-Flp)
Flp
Cre
IMPC Alleles IKMC - EUCOMM/KOMP
promoter loxP FRT
tm1d
See Skarnes et al. Nature, July 2011
BL/6N ES cells
(www.knockoutmouse.org)
www.mousephenotype.org
EUMODIC
Undertake a major pilot programme
Utilise standardised phenotyping pipeline
- EMPReSSslim
Analysis of 500 IKMC (EUCOMM) mutants
EU Framework Funded
4 Major Mouse Infrastructures
Assess the utility and efficiency of broad-based primary phenotyping of KO mice
Logistics of mouse production and phenotyping
Utility of assays (identifying disease models)
Sensitivity of assays (number of mice)
Capture, disseminate and build on rich phenotypic information
ICS
WTSI
HMGU
MRC
(www.eumodic.org)
www.mousephenotype.org (www.eumodic.org)
EUMODIC Workflow
Heterozygotes GLTs Homozygotes
Non-viable
homozygotes viable
homozygotes
Primary
Phenotyping
EMPReSSslim
Mouse clinics
Helmholtz, Munich
ICS, Strasbourg
MRC, Harwell
Sanger, Hinxton
7 males
7 females
www.mousephenotype.org
20 phenotyping platforms
406 phenotype parameters
155 metadata parameters
EMPReSSslim Primary Phenotyping Pipelines
www.mousephenotype.org
EUMODIC Summary:
500 lines committed to the pipeline => GLT or beyond
Data for 370 lines entered into EuroPhenome
All lines available through EMMA
Phenotyping finishes January 2012
(www.eumodic.org)
www.mousephenotype.org
EuroPhenome (www.europhenome.org)
www.mousephenotype.org
Rationale An Encyclopaedia of Mammalian Gene Function
Supporting a broad phenotyping effort would provide the following advantages:
A single cohort of mice would go through multiple phenotyping assays, so the cost of producing multiple cohorts in different laboratories for phenotyping would be eliminated.
Each mutant mouse strain would be characterized for a broad set of phenotypes in a way that will allow direct comparisons and result in a more thorough description of gene function.
Quality standards will be established and maintained, so the data will be of the highest reliability.
The risk of not finding a phenotype will be greatly reduced.
Important, but unpublishable, negative results will be captured.
www.mousephenotype.org
Future Vision An Encyclopaedia of Mammalian Gene Function
Build a resource of KO mice and associated encyclopedia of gene function, in a cost efficient and robust manner
Free thousands of researchers from tool generation
Uncover unforeseen novelty in mammalian gene function
A rich seam for future hypothesis driven research, with the potential for breakthrough discoveries
A transformative project that will underpin the future of biomedical science and the biology of disease systems
www.mousephenotype.org
IMPC Activities
Undertake broad based primary phenotyping of 20,000 mutants from the IKMC resource
A coordinated effort of mouse clinics worldwide
Phase I (2011-2016): phenotype up to 5,000 lines
Pipeline development, logistics
Phenotyping technology developments e.g. imaging
Ramp up
Phase II (2016-2021): Phenotype 15,000 mutants
Data freely available through a Data Coordination Centre, supported by R&D groups at clinics
www.mousephenotype.org www.mousephenotype.org
www.mousephenotype.org
www.mousephenotype.org
IMPC Engagement Building EXPERT Networks
Building up a PHENOMIN Mouse Networks to incorporate experts in:
Neuro
Obesity and Diabetes
Ubiquitination
Bone
Liver
Haematopoiesis
Fibrosis
Vision
Respiratory
Renal
Cardiovascular
Development
Immunology and infection
Research consortia
Individual labs
Privately held Companies
…
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www.mousephenotype.org
Challenges Ahead 1
Delivering a rich, robust phenotype pipeline that meets the needs of the community
Learning from each other
New assays/new disease areas
Development pipeline
Addressing the challenge of aging phenotypes – from cancer to neurodegeneration
Delivering to the consortium an effective data acquisition, data analysis and data dissemination pipeline
Statistical approaches to annotation
Development of approaches to describe and map phenotypes to human disease states
Working with the ontology community
www.mousephenotype.org
Challenges Ahead 2
Networking with the community
Ensuring utilisation of data and uptake of resources
Fostering networks of activity that add value and understanding
Capturing secondary and tertiary phenotyping information
Measuring and reporting that activity
Bringing the community into the fold
Incorporation of specialist centres into IMPC e.g. aging
Implementation of niche, challenge and sensitised screens
Integration with phenotyping of other genetic reference populations
Link-up with planning for phenotyping in the CC community, outbred studies
www.mousephenotype.org
EXCELLENCE in MOUSE FUNCTIONAL GENOM Please visit our new website http://www.ics-mci.fr/
ALI-HADJI Dalila DIERICH Andrée LEGEAY Sandrine RIO-ZENNER Fanny
AMANN Grégory DREYER Dominique LEYRITZ Johan ROTH Christelle
ANDRE Philippe EGLINGER Yolande LINDNER Loic ROUSSEAU Stéphane
AUBURTIN Aurélie EL FERTAK Leila LORENTZ Romain ROUSSEAU Valérie
AUGE Fabrice EL FERTAK Lahcen LUPPI Laurence SCHMITT Raphaël
AYADI Abdelkader ENNAH Hamid LUX Aline SCHOEDEL Christophe
BAM'HAMED Chaouki ERBS Valérie MELLUL Peggy SCHWOERER Marie-Jeanne
BANQUART- OTT Nadine ESSABRI Karim MERTZ Annelyse SEITZ Thierry
BECKER Julien FISCHER Fabienne MEZIANE Hamid SELLOUM Mohammed
BEDU Elodie FISCHER Natacha MITTELHAEUSER Christophe SORG-GUSS Tania
BIRLING Marie-Christine FOUGEROLLE Jean-Victor MOKNI Mourad SUTTER WOLTER Anne
BLONDELLE Eric FRICKER Bastien MONTIAL Marina TILLY Isabelle
BOUR Raphaël GOETZ-REINER Patrice MORO Anne-Isabelle TOUBARI Chadia
BRIGNON Sophie GONCALVES DA CRUZ Isabelle MOULAERT David UZUN Ibrahim
BUNZ Isabel GRUBER Frédéric MULLER Stéphanie VASSEUR Laurent
CARADEC Claudia GUIMOND Alain NAGRE Isabelle VENTEO Lydie
CAYROU Pauline HERAULT Yann OHLMANN Thierry VINCENT Cindy
CES Aurélia HEMMERLE Mathieu PAVLOVIC Guillaume WAGNER Christel
CHAMPY Marie-France JACQUOT Sylvie PENSAVALLE Joëlle WALCH Laëtitia
CHARLES Philippe JAGLA-EBERLIN Monika PERRY Mélanie WALLERICH Sandrine
CHARTOIRE Nathalie KUJATH Christelle PETER Emilie WEBER Bruno
CHEBBOUB Djaouida KURTZ Caroline PETIT-DEMOULIERE Benoît WENDLING Olivia
CLAEYSSEN Richard LAEUFER Laurent PHAM Thi Bich Hanh WETZSTEIN Eric
COMBE Roy LALANNE Valérie POUILLY Laurent WIECROCK Cyrille
DEBOUZY Guillaume LE MARCHAND Elise QUEUCHE Danielle WIECROCK Ludovic
DELANGLE Benoît LEBLANC Sophie RIET Fabrice ZANINELLO Fabienne
LECOCQ Muriel ZANINELLO Nathalie