A Highly Efficient Method For GPCR Gene-targeted Vector Construction By Red Recombination System

G protein coupled receptor family (GPCR) is consisted of a polypeptide chain containing7transmembrane domains. It accounts for3%-4%of the human genome. GPCR has widely physiological effects and it is one of the most popular drug targets. Now the biological function and ligand of most Adhesion-GPCRs is still unknown. so it is also named orphan receptor. A study on the biological function of Adhesion-GPCRs has great significance in basic theoretical research and drug potential target discovery research.Phylogenetic analysis of GPCRs shows that the Adhesion-GPCRs is an unique family. Adhesion-GPCRs are characterised by long N termini.The N termini domain are often consist of a range of protein domains found in cell adhesion proteins. Obviously the study for Adhesion-GPCRs is required a lot of work to do. So we constructed the gene-targeting vector of Gpr56, Bail, Gpr111and Gpr126by using our system. Using the vectors mentioned above, we can study those genes at the molecular genetic level based on the knockout mouse model.At present, based on the knockout mouse model to study the function of gene has become an important academic field. However, construction of targeting vector remains a time-consuming and technically challenging process with the traditional method of constructed targeting vector. Here we describe a highly efficient recombineering-based method by red recombination system. In this way, we can construct targeting vector highly efficient through two times of recombination. In addition, we improved the method of conditional targeting constructoion by red recombination system. The two LoxP sites can be more accurate inserted into the target position with this improved method.