title: N-ethyl-N-nitrosourea-based generation of mouse models for mutant G protein-coupled receptors.
abstract: Chemical random mutagenesis techniques with the germ line supermutagen N-ethyl-N-nitrosourea (ENU) have been established to provide comprehensive collections of mouse models, which were then mined and analyzed in phenotype-driven studies. Here, we applied ENU mutagenesis in a high-throughput fashion for a gene-driven identification of new mutations. Selected members of the large superfamily of G protein-coupled receptors (GPCR), melanocortin type 3 (Mc3r) and type 4 (Mc4r) receptors, and the orphan chemoattractant receptor GPR33, were used as model targets to prove the feasibility of this approach. Parallel archives of DNA and sperm from mice mutagenized with ENU were screened for mutations in these GPCR, and in vitro assays served as a preselection step before in vitro fertilization was performed to generate the appropriate mouse model. For example, mouse models for inherited obesity were established by selecting fully or partially inactivating mutations in Mc4r. Our technology described herein has the potential to provide mouse models for a GPCR dysfunction of choice within <4 mo and can be extended to other gene classes of interest.
authors.affiliation: Ingenium Pharmaceuticals AG, Martinsried, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Grosse
authors.forename: Patrick
authors.initials: P
authors.lastname: Tarnow
authors.forename: Holger
authors.initials: H
authors.lastname: Römpler
authors.forename: Boris
authors.initials: B
authors.lastname: Schneider
authors.forename: Reinhard
authors.initials: R
authors.lastname: Sedlmeier
authors.forename: Ulrike
authors.initials: U
authors.lastname: Huffstadt
authors.forename: Dirk
authors.initials: D
authors.lastname: Korthaus
authors.forename: Michael
authors.initials: M
authors.lastname: Nehls
authors.forename: Sigrid
authors.initials: S
authors.lastname: Wattler
authors.forename: Torsten
authors.initials: T
authors.lastname: Schöneberg
authors.forename: Heike
authors.initials: H
authors.lastname: Biebermann
authors.forename: Martin
authors.initials: M
authors.lastname: Augustin
mesh_terms: D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis / Q000379:methods; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005038:Ethylnitrosourea / Q000633:toxicity*; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D016296:Mutagenesis / Q000187:drug effects; D009154:Mutation / Q000235:genetics*; D010802:Phylogeny; D044104:Receptor, Melanocortin, Type 3 / Q000235:genetics / Q000502:physiology; D044105:Receptor, Melanocortin, Type 4 / Q000235:genetics / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
chemical_list: D000477:Alkylating Agents; D044104:Receptor, Melanocortin, Type 3; D044105:Receptor, Melanocortin, Type 4; D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins; D005038:Ethylnitrosourea