Institut Clinique de la Souris

Yann Herault CNRS, INSERM, Université de Strasbourg,

1 rue Laurent Fries - 67404 Illkirch, France. herault@igbmc.fr

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

Department for Phenotyping

7

Head T. Sorg

7

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

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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