Detection Of Genetically MC4R Gene Copy Number And Integration Sites

Melanocortin receptor4(Melanocortin receptor4,MC4R) gene is one of the major gene affecting growing and finishing pigs performance,is involved in the regulation of body weight,feed intake and energy balance of the key signal substances. Transgenic animal technology is mainly used in the production of valuable proteins, gene therapy,organ transplantation,improved varieties and animal disease models and other aspects of the establishment,the current people have gained such as sheep,cattle,mice,pigs and other animals transgenic cloned in our laboratory also received a transfer MC4R cloned pigs in2013.Determine the copy number and integration sites of the foreign gene is an exogenous gene for subsequent phenotypic and functional studies investigate prerequisite.Therefore,this experiment will MC4R gene into pig PK15cells cultured pick monoclonal,thereby simulate transgenic cloned pigs through three pairs of specific primers for PCR amplification of transfected cells do positive cells detected by real-time quantitative PCR detection of gene copy number abroad,ligation-mediated PCR to detect transgene integration sites abroad,initially established a foreign gene copy number,experimental system integration testing sites.The main results are as follows:(1) Use ligation-mediated PCR to detect porcine MC4R gene into the mouse genome on6chromosome No. of37236325-37236754range place.(2) Successfully constructed pig pcDNA3.1(+)-MC4R expression plasmid, and transferred to pig PK15cells obtained monoclonal cell34.(3) Pick made34monoclonal cells by PCR,16positive cells,transfection efficiency was47%.(4) By real-time PCR detection of copy number16positive cells was1.42084MC4R gene was constructed experimental system using real-time quantitative PCR exogenous gene copy number.(5) By ligation-mediated PCR successfully3monoclonal cell integrated exogenous gene locus on chromosome14pigs at115863630-115863923established using ligation-mediated PCR detection of the site of integration of exogenous genes experimental system.