Construction Of Tetracycline-inducible Pig MC4R Eukaryotic Expression Vector And Its Expression In Vitro

MC4R is a class of peptide secreted by the hypothalamic ventromedial nucleus and belongs tothe family of seven transmembrane G protein coupled receptors·The pig MC4R gene is consideredto be one of the major genes affecting on the growth and fatness traits in pigs, and it was a keysignaling substances that regulate body weight,feeding and the balance of energy,therefore it isgreat significance to research gene function and KEGG pathways of MC4R gene. However, in theprocess of study eukaryotic gene function, sustained expression of the exogenous gene becomesinterested in the study of gene function has great limitations. The emergence oftetracycline-inducible expression system to overcome this shortcoming, it has become the mostwidely used inducible expression system, regulation of gene expression in time, a more detailedstudy of gene function..This experiment used the Tet-On Gene Expression System,it is constituted by thetranscription element,the reaction element and inducer. By using the molecular biology technology,cloned sequence of pig MC4R gene, pig MC4R gene was inserted into pTRE vector downstreamMCS sites,in order to build pTRE-MC4R（P1）vectort. Cloning the main part of transcriptioncomponents and reaction element, pig MC4R gene was inserted into MCS sites of the reactioncomponents, the above-mentioned components were linked into pcDNA3.1（+）to constructrecombinant pcDNA3.1（+）-rtTA-PTight-MC4R（P2）vector by double digestion and connectionsmethods. The recombinant plasmid P1and P2were cotransfected into pig fibroblasts with Dox at5ug/mL, detect MC4R gene expression and MC4R gene-related expression changes in before andafter induction.To prepare for further study the biological function of the MC4R gene andpreparation of high survival rate of transgenic pigs.The main results are as follows：1） Have exactly cloned the Complete sequence of cDNA of MC4R gene,the size of999bp.2） Have successfully built P1and P2inducible expression vector expressing pig MC4R gene.3） P1and the pTet-On vector were cotransfected into fibroblasts cells with Dox,MC4R geneexpression was significantly increased (P <0.01).4） The fusion vector P2was cotransfected into fibroblasts cells with Dox, the same of MC4Rgene expression was significantly increased (P <0.01).5） Inducibled expression MC4R gene by recombinant plasmid P2was cotransfected,POMCgene expression was significantly increased (P <0.05), and the MC3R, AGRP, NPY, POMC, Leptin gene expression levels did not change significantly.