Systematic generation of in vivo G protein-coupled receptor mutants in the rat

Hubrecht Institute for Developmental Biology and Stem Cell Research, Cancer Genomics Center, Royal Netherlands Academy of Sciences and University Medical Center Utrecht, Uppsalalaan 8, Utrecht, The Netherlands.
The Pharmacogenomics Journal (Impact Factor: 4.23). 10/2011; 11(5):326-36. DOI: 10.1038/tpj.2010.44
Source: PubMed


G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies.

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    • "Adult male Wild-Type (WT) and MC4R Knockout rats (MC4RKO) generated via targeted chemical mutagenesis at the Hubrecht Institute in the lab of Dr. Edwin Cuppen (Van Boxtel et al. 2011) and licensed and marketed by Transposagen/Taconic were acquired at 10 weeks of age and studied up to 20 weeks of age. All animals were maintained in an AAALAC-approved animal facility and the GHSU Institutional Animal Care and Use Committee approved all procedures. "
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