GENERATION of CRE‐ERT2 TRANSGENIC MOUSE LINES FOR TIME AND CELL SPECIFIC CONDITIONAL GENE INACTIVATION Marie‐Christine Birling Institut Clinique de la Souris, Strasbourg 1 Why a creERT2 resource? 1. Inactivation of the 2 copies is lethal for 33% of the genes 2 Why a creERT2 resource? 1. Inactivation of the 2 copies is lethal for 33% of the genes 2. Study the function of a gene 1. In a specific organ/ cell populations 2. In the adult 3 The ICS CreERT2 zoo o 43 lines characterized or under characterization http://www.ics‐mci.fr/mousecre 4 A standardized characterization (i) Line generated by pronuclear injection (PNI) of mainly BAC – creERT2 5 ∼250 lines; 50 projects Specificity of Cre expression (RT‐qPCR) 1. RT‐qPCR high throughput screen Specificity of endogenous gene expression (RT‐qPCR) 2. Extensive analysis of best line Cre activity at the anatomic level (lacZ staining) A standardized characterization (ii) Line generated by pronuclear injection (mainly BAC transgenes) ∼250 lines; 50 projects 1. RT‐qPCR high throughput screen 2. Extensive analysis of best line For some projects 3. Additional characterization 4. Publications 6 Ins1‐creERT2 Specificity of Cre expression (RT‐qPCR) High level in pancreas Low level in brain 7 Ins1‐creERT2 Ins1‐creERT2 x Rosa26‐ loxSTOPlox‐LacZ Female 60 (tam) IHC Cre Abcam antibody x40 LacZ staining x10 8 Female 144 (tam) Male 35 (oil) Ins1‐creERT2 After Tamoxifen injection IHC cre antibody Pancreas ‐ Islets of Langerhans triple IHC staining Somatostatin (purple) Cre protein (brown) lacZ staining on Rosa26 reporter line 9 Glucagon (pink) Ins1‐creERT2 Fact: the Rat Insulin Promoter (RIP)‐Cre mice display glucose intolerance (Lee et al., 2006) Glucose tolerance test under chow diet Intraperitoneal injection of sterile‐filtered D‐glucose (2g/kg of fasted body weight) Rat insulin I Promoter‐ Cre Rat insulin I Promoter‐ Cre C57BL/6 Published data C57BL/6 RIP‐Cre C57BL/6 ICS data Question : Is the Ins1‐CreERT2 line glucose intolerant? 10 Lee et al., “RIP‐Cre revisited, evidence for impairments of pancreatic b‐cell function.”J Biol Chem, 2006 Ins1‐creERT2 Answer: No! Glucose tolerance test under chow diet No effect of the construction in glucose tolerance test 11 Note: We observe no relevant difference between Ins1‐creERT2 mice and WT mice treated with the vehicle Ins1‐creERT2 Conclusion Efficiency of Cre expression and Cre mediated excision is highly specific Brain: lacZ is negative but is Soriano Rosa26Neo‐lacZ a good reporter ? The Ins1‐creERT2 line seems specific to β‐cells Our line does not present the RIP‐cre phenotype under chow diet There is no diabetes onset in Ins1‐creERT2 mice treated or not with tamoxifen => The Ins1‐CreERT2 model is validated 12 Gcg‐creERT2 Plasmid transgene Cre expression RT-qPCR Additional characterization: Immunohistochemistry Female 13 Male Testis Pancreas Liver Duodenum Ovary Pancreas Liver Duodenum Relative level Cre antibody Glucagon antibody Male / tam injection Ela1‐creERT2 chymotrypsin-like elastase family, member 1 BAC transgene Cre expression Endogenous gene (RT‐qPCR) Symatlas data 14 Ela1‐creERT2 Additional characterization: Cre immunohistochemistry Tam injection Pancreas x 40 (Novagen antibody) Wax embedded tissues 15 Ins1‐creERT2 Gcg‐creERT2 TAM Ela1‐creERT2 TAM => An inducible Cre line for 3 cell types of the mouse pancreas 16 TAM A comparative study Ubiquitous CreERT2 lines Characterization by breeding with the Soriano Rosa26Neo‐lacZ reporter: Cre activity at the anatomical level 17 PPM1a‐CreERT2 (BAC transgene) Rosa‐CreERT2 (Hameyer et al., 2007) Rosa‐ERT2‐iCre‐ERT2 (Jullien et al., 2008) A comparative study: Ubiquitous CreERT2 lines Specificity of Cre expression (RT‐qPCR) 18 Ubiquitous creERT2 lines Efficiency of Cre mediated excision at the cellular level (breeding by the Rosa26Neo‐lacZ reporter) Aorta Heart Liver Pancreas Colon Kidney Skin Testis Ppm1a‐ creERT2 Aorta :+ Adipose tissue: ++ Ventricle: + +++(+) + (patchy/ mosaic) Muscularis propria : + Mucosa : + (patchy/mosaic) Cortical :+++ Medullar:+++ Papilla:++++ Epidermis:+++ Basal cells:++ Dermis:+ Hair follicle:+++ Sebaceous gland:+ Seminiferous tubule: ++ Rosa26‐ creERT2 Aorta :‐/+ Adipose tissue: ‐/+ Ventricle: + +++(+) + (patchy/ mosaic) Muscularis propria : ‐ Mucosa : ‐/+ Cortical :+ Medullar:+(+) Papilla:+++ Epidermis:+++ Basal cells:++ Dermis:‐ Hair follicle:+++ Sebaceous gland:+ Seminiferous tubule: + ERT2‐cre‐ ERT2 Aorta:+ Adipose tissue:+ Ventricle: +/‐ +++ + (patchy/ mosaic) Muscularis propria : + Mucosa : + (patchy/mosaic) Cortical :‐/+ Medullar:+ Papilla:++ Epidermis:++ Basal cells:+ Dermis:+ Hair follicle:++ Sebaceous gland:+ Seminiferous tubule: ++ 19 Ppm1a creERT2 line IHC with anti‐Ppm1a and anti‐Cre after Tamoxifen induction Ppm1a‐CreERT2 activity avec breeding with Rosa26Neo‐lacZ Skin Liver Muscle IHC Ppm1a IHC Cre (Novagen antibody) Good co‐localization of the CreERT2 with the endogenous protein LacZ staining on Ppm1a‐CreERT2 X Rosa Neo‐Lac 20 Lydie Venteo Ppm1a creERT2 line Conclusion The Ppm1a‐CreERT2 line is at least as efficient as the Rosa‐ CreERT2 and Rosa‐ERT2‐iCre‐ERT2 lines after Tamoxifen induction in the adult mouse. •Globally higher mRNA expression •Better excision rate on the Rosa Neo‐LacZ reporter line •Good co‐localization of the CreERT2 protein with the endogenous Ppm1a protein •No leaking (no Cre mediated excision) observed in the absence of Tamoxifen 21 Myh6‐CreERT2 ‐myosin, heavy polypeptide 6, cardiac muscle, alpha ‐BAC construct ‐cardiac muscular cells Specificity of Cre expression Endogenous gene (RT‐qPCR) 22 Myh6‐CreERT2 http://www.ics-mci.fr/mousecre/results/histo?subline_id=252 23 Adipoq‐CreERT2 adiponectin, C1Q and collagen domain containing Human BAC transgene Specificity of Cre expression Endogenous gene (RT‐qPCR) 24 Adipoq‐CreERT2 Line validated, very little leaking observed 25 The flow scheme Line generated by pronuclear injection (PNI) of a BAC – creERT2 ∼250 lines; 50 projects 1. RT‐qPCR high throughput screen 2. Extensive analysis of best line 3. Database 4. distribution (EMMA) 26 4. Distribution following email contact: [email protected] Please visit our new website http://www.ics‐mci.fr/ A CENTRE OF EXCELLENCE FOR MOUSE FUNCTIONAL GENOMICS HERAULT Yann ALI‐HADJI Dalila AMANN Grégory ANDRE Philippe AUBURTIN Aurélie AUGE Fabrice AYADI Abdelkader BAM'HAMED Chaouki BANQUART‐OTT Nadine BECKERJulien BEDU Elodie BIRLING Marie‐ Christine BLONDELLE Eric BOUR Raphaël BRIGNON Sophie BUNZ Isabel CARADEC Claudia 27 CAYROU Pauline CES Aurélia CHAMPY Marie‐France CHARLES Philippe CHARTOIRE Nathalie CHEBBOUB Djaouida COMBE Roy DEBOUZY Guillaume DELANGLE Benoît DIERICH Andrée DING Thomas DREYER Dominique EL FERTAK Leila EL FERTAK Lahcen ENNAH Hamid ERBS Valérie FOUGEROLLE Jean‐ Victor GOETZ REINER Patrice GONCALVES DA CRUZ ‐ Isabelle GRUBER Frédéric GUIMOND Alain HEMMERLE Mathieu JACQUOT Sylvie KUJATH Christelle KURTZ Caroline LAEUFER Laurent LE MARCHAND Elise LEBLANC Sophie LECOCQ Muriel LEGEAY Sandrine LINDNER Loïc LORENTZ Romain LUPPI Laurence MELLUL Peggy MERTZ Annelyse MEZIANE Hamid MITTELHAEUSER Christophe MOKNI Mourad MONTIAL Marina MORO Anne‐Isabelle NAGRE Isabelle OHLMANN Thierry PAVLOVIC Guillaume PENSAVALLE Joëlle PETER Emilie PETIT‐DEMOULIERE Benoît PHAM THI BICH Hanh POUILLY Laurent QUEUCHE Danielle RIET Fabrice RIO‐ZENNER Fanny ROTH Christelle ROUSSEAU Valérie ROUSSEAU Stéphane SCHMITT Raphaël SCHWOERER Marie‐ Jeanne SEITZ Thierry SELLOUM Mohammed SORG‐GUSS Tania 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 WOLTER Anne ZANINELLO Fabienne ZANINELLO Nathalie
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