N-ethyl-N-nitrosourea-based generation of mouse models for mutant G protein-coupled receptors

Physiol Genomics. 2006 Aug 16;26(3):209-17. doi: 10.1152/physiolgenomics.00289.2005. Epub 2006 May 23.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkylating Agents / toxicity
  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • DNA Mutational Analysis / methods
  • Disease Models, Animal*
  • Enzyme-Linked Immunosorbent Assay
  • Ethylnitrosourea / toxicity*
  • Female
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Mice
  • Mice, Inbred C3H
  • Mice, Inbred C57BL
  • Mice, Mutant Strains
  • Mutagenesis / drug effects
  • Mutation / genetics*
  • Phylogeny
  • Receptor, Melanocortin, Type 3 / genetics
  • Receptor, Melanocortin, Type 3 / physiology
  • Receptor, Melanocortin, Type 4 / genetics
  • Receptor, Melanocortin, Type 4 / physiology
  • Receptors, G-Protein-Coupled / genetics*
  • Receptors, G-Protein-Coupled / physiology
  • Signal Transduction / physiology
  • Transfection

Substances

  • Alkylating Agents
  • Receptor, Melanocortin, Type 3
  • Receptor, Melanocortin, Type 4
  • Receptors, G-Protein-Coupled
  • Green Fluorescent Proteins
  • Ethylnitrosourea