Institut Clinique de la Souris
The French Contribution to the IMPC
Yann Herault CNRS, INSERM, Université de Strasbourg,
1 rue Laurent Fries - 67404 Illkirch, France. [email protected]
The French National Infrastructure for Mouse PHENOGENOMICS
M. Selloum, M.-C. Birling C. Olivier, S. Lerondel F. Fiore, B. Malissen, G. Pavlovic, A. Ayadi, P. Lopes, C. Fremond, T. Sorg, Y. Herault
TAAM
ICS CIPHE
WP1: Management
USE
RS/
PAR
TNER
S
WP2
Mutagenesis WP3
Zootechny WP4
Phenotyping
USE
RS/
PAR
TNER
S
WP5: Discover of mammalian genes function /
Contribution to IMPC
1. ES Cells to Mouse 180 40 30
2. Mouse to Cohort 180 40 30
3. Cohort to Phenotype 250 250
4. Archive and Distribute 180 40 30
5. Tech and Tools Dev 1,2,3 4 1,3
promoter
start codon
stop codon
prom-Neo
FRT loxP FRT loxP loxP SA-lacZ-pA
Cre recombinase cre
promoter
start codon
stop codon
loxP FRT SA-lacZ-pA
lacZ
Critical exon
tm1a
tm1b (post-Cre)
KO first allele (reporter-tagged insertion allele)
QC and procedures from ES to cohorts
•• southsouthern ern blot blot anaanalylysis sis ffor ror rececombinaombination tion ccononfirmafirmation tion as as wwellell asasccononfirmafirmation tion of tof the he prpresence esence of the of the 3’ 3’ llooxPxP sitsite,e,
•• kkararyyotyotyping of 2 vping of 2 validaalidatted ed clonesclones•• micrmicroo--injection injection in 50 in 50 BBALB/ALB/cNcN blasblasttocyocytteses•• esesttablishablishmemennt t of of chimerchimeraass•• brbreeding eeding of the of the 4 bes4 best t chimerchimeras as ((chimerichimerismsm >45%)>45%)•• GLGLT assessT assessmemennt t (tm1a(tm1a allelallelees s ffor or crcryyoo))•• GenerGeneraation tion of tm1b of tm1b KKO allele O allele (pos(postt--CrCree))•• IVF IVF quick quick eexpansionxpansion HeHet t (4 males and 8 females)•• HeHet t x Hex Het t crcrossoss HomHom (4 males and 4 females)•• HomHom X HeX Het t crcrosses: osses: prproducoduction oftion of ccohoohort, frt, fertiliertility ty and and viviabiliabilityty
ttesests ts (n=4)(n=4 Males and females)•• 1 Cohort 1 Cohort of cof conontrtrol per wol per weekeek
Genetic Tools Dev.: Speeding up the Cre-Step with Fluo marker
a b
c d
Rosa26Cre-GFP/0
Rosa26Flp-YPF/0
B6N
M-C. Birling, G. Pavlovic
Cre eGFP Neo 2A
FlpO eYFP pA Neo 2A pCAGGS
pChick b-act
Rosa26 WT
Rosa26Cre-GFP(FRT-Neo-FRT)
Rosa26FLP-YFP(LoxP-Neo-LoxP)
pA
PCR1
PCR4 PCR2
Cre eGFP 2A pChick b-act pA
PCR4 PCR3
Rosa26Cre-GFP(FRT)
PCR5 PCR7
FlpO eYFP pA
CR7
2A
P
Maternal Rosa26 allele
WT Flp-YFP(loxP-Neo-loxP) P
ater
nal
Ro
sa2
6
alle
les
WT
Cre-GFP (FRT-Neo-FRT)
WT/WT
14
Maternal contribution (FRT)/Cre-GFP WT
10
WT/Flp-YFP(LoxP-Neo-loxP)
13
Cre-GFP (FRT) /Flp-YFP(loxP)
7
Maternal Rosa26 allele
WT Cre-GFP (FRT-Neo-FRT)
Pate
rnal
Ro
sa2
6
alle
les WT
WT/WT
29
WT/Cre-GFP(FRT-Neo-FRT)
28
Flp-YFP (LoxP-Neo-loxP)
Maternal contribution
Flp-YFP (loxP)/WT
28
Flp-YFP(loxP)/Cre-GFP (FRT)
13
pCAGGS
Rosa26FLP-YFP(LoxP)
PCR6
Genetic Tools Dev.: and Maternal Cre effect Rosa26Cre-GFP(FRT)-/Flp-YFP(LoxP)
1 2 3 4 5 6 7
Rosa26Cre-GFP(FRT-Neo-FRT)/ WT
1 2 3 4 PCR
LoxP FRT
M-C. Birling, G. Pavlovic
16
9 13 10 11 14 15 12
Weight Curve – 5wk to 16wk
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In Bo
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Multiplex analysis 7 M + 7 F Mutant Adult Mice
e re
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ired
ht g n
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y
We
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TECH DEVELOPMENT Cardiovascular
Behavior and cognition Nutrition, metabolism
Neuromuscular system
Embryology and Reproduction
Immunology and Inflammation
Respiration, allergy Imaging immune responses to pathogens
Skeleton, Tooth and Craniofacial morphology
Cytometry by time-of-flight mass spectrometry Electrophysiology
Scientific Experts: Block-Zupan A., Chambon P., Chan S., Fiore F., Frossard N., Gorvel J.-P., Grenot P., Hérault Y., Kieffer B., Laporte J., Lepape A., 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., …
Follow gestation by micro-ultrasonography
at E5 and E8 (count the deciduas)
No or few (less than 40 %) resorptions
Collect fetuses at E18.5
Externally malformed fetuses
Fix 3 litters in Bouin’s solution
No mutant detected
Collect embryos at E10.5
Externally normal fetuses
Fix 3 litters in Klotz’s solution
No mutant detected
Collect embryos at E10.5
Genotyping
Mutants detected
Autopsy (Klotz) or Serial sections (Bouin)
High level of resorptions
Collect embryos at E10.5
Fix 2 litters in paraformaldehyde
b-galactosidase expression by optical
tomography
Genotyping
No mutant detected
End of analysis (Death must occur around implantation or gastrulation)
Fix 2 litters in Bouin’s fluid
Mutants detected
Serial sections
Gestation at E7
44/105
O. Wendling, M. Mark
TECH DEVELOPMENT Cardiovascular
Behavior and cognition Nutrition, metabolism
Neuromuscular system
Embryology and Reproduction
Immunology and Inflammation
Respiration, allergy Imaging immune responses to pathogens
Skeleton, Tooth and Craniofacial morphology
Cytometry by time-of-flight mass spectrometry Electrophysiology
Scientific Experts: Block-Zupan A., Chambon P., Chan S., Fiore F., Frossard N., Gorvel J.-P., Grenot P., Hérault Y., Kieffer B., 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., …
Landmark and methodologies for craniofacial analysis
L. Viriot, P. Lopes, E. Velot, S. Lerondel, Y. Herault
TECH DEVELOPMENT Cardiovascular
Behavior and cognition Nutrition, metabolism
Neuromuscular system
Embryology and Reproduction
Immunology and Inflammation
Respiration, allergy Imaging immune responses to pathogens
Skeleton, Tooth and Craniofacial morphology
Cytometry by time-of-flight mass spectrometry Electrophysiology
Scientific Experts: Block-Zupan A., Chambon P., Chan S., Fiore F., Frossard N., Gorvel J.-P., Grenot P., Hérault Y., Kieffer B., Laporte J., Lepape A., 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., …
Lipopolysaccharide (LPS) challenge
0,00
2,00
4,00
6,00
8,00
10,00
0 100 200 300 400 500
ctrl
LPS 1µg
LPS 5µg
LPS 10 µg
BALF Serum BALF Serum
Pulmonary and inflammatory Function (6h) E. Dalloneau, J. Becker, Y. Herault
Lipopolysaccharide (LPS) challenge
Prmt2 NF-kB
TLR4
TNFα IL-6
LPS
0
1
2
3
4
5
6
52
03
55
06
58
09
51
10
12
51
40
15
51
70
18
52
00
21
52
30
24
52
60
27
52
90
30
53
20
33
5 min
Pulmonary airway resistance WT NaCl
Prmt2+/- NaCl
WT LPS
Prmt2+/- LPS
0
200
400
600
800
1000
pg/
ml
[TNF] **
**
NaCl LPS
E. Dalloneau, J. Becker, Y. Herault (J. Immunol in press)
0
400
800
1200
1600
2000
pg
/ml
[IL-6] **
**
NaCl LPS
TECH DEVELOPMENT Cardiovascular
Behavior and cognition Nutrition, metabolism
Neuromuscular system
Embryology and Reproduction
Immunology and Inflammation
Respiration, allergy Imaging immune responses to pathogens
Skeleton, Tooth and Craniofacial morphology
Cytometry by time-of-flight mass spectrometry Electrophysiology
Scientific Experts: Block-Zupan A., Chambon P., Chan S., Fiore F., Frossard N., Gorvel J.-P., Grenot P., Hérault Y., Kieffer B., Laporte J., Lepape A., 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., …
Profiling of the mouse innate and adaptive immune system under normal and confined
(BSL3) conditions via the quantitative analysis of over 200 parameters.
Mass Cytometer
INSERM US12
Standard operating procedure for quantitative analysis of the lymphoid and myeloid cells
present in small blood samples.
Standard operating procedure for
quantitative analysis of dendritic cells.
REACHING THE COMMUNITY > 1000 Lab Users
CIPHE ICS
TAAM
European funded networks
21
1953-2011: 58 years serving the scientific community
BERTEAUX LIONEL
BERTRAND MARIE-FRANCOISE
BILAN Christiane
BORNAT ANGELIQUE
BOULANGER VIRGINIE
CHAVANEAU GHISLAINE
CHERFOUH AMEZIANE
CLEMENCON RUDY
DESALE ELODIE
DESSAIN MARIE LAURE
DHENNIN LAURENT
DHENNIN RAPHAELLE
DIET Alexandre
DOUIN Estelle
DUFOUR ALAIN
DURAND TAMARA
FOURNIER KARINE
FREMOND CECILE
FROUX NATHALIE
HERPIN Alexandre
JAMBOU KARINE
JOSEPH Laurent
JOUSSARD GUILLAUME
LANDREAU CATHERINE
LARRIGALDIE VANESSA
LE BRIS KHEIRA
LE MEE MARYLINE
LERONDEL STEPHANIE
LORY RACHEL
MAILLARD Karine
MAISTRE Jean-baptiste
MARTIN Christelle
MAUNY PASCAL
MAURIO AGNES
MEAUXSOONE JOEL
MEDINA PATRICE
MERCIER LUDOVIC
MOTHU Nadya
MOUSSU JEAN-PAUL
PAPION DUCHATEAU RENE
PATURANCE SEBASTIEN
PERES PHILIPPE
RENIER ANNICK
RETIF LEROUX STEPHANIE
SEGHROUCHNI EMMANUELLE
SENECHAL ARMELLE
THERET YVETTE
THOMAS Jean-Luc
TORSET MONIQUE
VOISIN OLIVIER
ZIADI ABDELMALEK
LERONDEL STEPHANIE
LORY RACHEL
MAILLARD Karine
http://transgenose.cnrs-orleans.fr/taam/presentation.php
Please visit our new website http://www.ics-mci.fr/
ALI-HADJI Dalila
AMANN Grégory
ANDRE Philippe
AUBURTIN Aurélie
AUGE Fabrice
AYADI Abdelkader
BAM'HAMED Chaouki
BANQUART- OTT Nadine
BECKER Julien
BEDU Elodie
BIRLING Marie-Christine
BLONDELLE Eric
BOUR Raphaël
BRIGNON Sophie
BUNZ Isabel
CARADEC Claudia
CAYROU Pauline
CES Aurélia
CHAMPY Marie-France
CHARLES Philippe
CHARTOIRE Nathalie
CHEBBOUB Djaouida
CLAEYSSEN Richard
COMBE Roy
DEBOUZY Guillaume
DELANGLE Benoît
DIERICH Andrée
DREYER Dominique
EGLINGER Yolande
EL FERTAK Leila
EL FERTAK Lahcen
ENNAH Hamid
ERBS Valérie
ESSABRI Karim
FISCHER Fabienne
FISCHER Natacha
FOUGEROLLE Jean-Victor
FRICKER Bastien
GOETZ-REINER Patrice
GONCALVES DA CRUZ Isabelle
GRUBER Frédéric
GUIMOND Alain
HERAULT Yann
HEMMERLE Mathieu
JACQUOT Sylvie
JAGLA-EBERLIN Monika
KUJATH Christelle
KURTZ Caroline
LAEUFER Laurent
LALANNE Valérie
LE MARCHAND Elise
LEBLANC Sophie
LECOCQ Muriel
LEGEAY Sandrine
LEYRITZ Johan
LINDNER Loic
LORENTZ Romain
LUPPI Laurence
LUX Aline
MELLUL Peggy
MERTZ Annelyse
MEZIANE Hamid
MITTELHAEUSER Christophe
MOKNI Mourad
MONTIAL Marina
MORO Anne-Isabelle
MOULAERT David
MULLER Stéphanie
NAGRE Isabelle
OHLMANN Thierry
PAVLOVIC Guillaume
PENSAVALLE Joëlle
PERRY Mélanie
PETER Emilie
PETIT-DEMOULIERE Benoît
PHAM Thi Bich Hanh
POUILLY Laurent
QUEUCHE Danielle
RIET Fabrice
RIO-ZENNER Fanny
ROTH Christelle
ROUSSEAU Stéphane
ROUSSEAU Valérie
SCHMITT Raphaël
SCHOEDEL Christophe
SCHWOERER Marie-Jeanne
SEITZ Thierry
SELLOUM Mohammed
SORG-GUSS Tania
SUTTER WOLTER Anne
TILLY Isabelle
TOUBARI Chadia
UZUN Ibrahim
VASSEUR Laurent
VENTEO Lydie
VINCENT Cindy
WAGNER Christel
WALCH Laëtitia
WALLERICH Sandrine
WEBER Bruno
WENDLING Olivia
WETZSTEIN Eric
WIECROCK Cyrille
WIECROCK Ludovic
ZANINELLO Fabienne
ZANINELLO Nathalie