Generation Of MC3R Knock-out Pig By CRISPR/Cas9 System

The melanocortin receptors(MCRs)are a family of G protein-coupled receptors.So far,a total of five members have been found.MCRs are activated by melanocortin ligands and cascade the signals through increasing cAMP in the cells.Different members of MCRs were expressed in varietal cell types and played variance functions.MC3R gene high express in the hypothalamus,it was proved that MC3R play an important role in the regulation of animal appetite and energy homeostasis,but the mechanism was not clear.MC3R as candidate gene in hunman obesity syndrome,can provide new ideas and methods for the treatment of obesity by more in-depth study.Food intake and energy homeostasis affect pig economic traits,such as growth rate and lean percent of carcass.But MC3R knockout pigs are not yet reported.MC3R deficiency model was necessary not only for its functional study,but also for evaluation of its role in pig breeding,and for the treatment of obesity.CRISPR/Cas9 system,known as a new technology for genome editing,was used in this study.12 bp upstream of start codon,45 bp downstream of stop codon and other two site in the CDS of MC3R were chosen as four target sites and four vectors of MC3R-pX330 were constructed according to MC3R sequence information of Chinese Experimental Mini pig.Four vectors cut the target sites efficiently and specifically in primary culture of pig fibroblast.We got 10 MC3R homozygous knockout clones and 11 miscellaneous zygote clones in total of 72 clones,in which the positive rate is 29.16%.The pig fetal fibroblasts were transfected with constructed MC3R-pX330 knockout vector by electroporation.Furthermore,we also got 1 MC3R knockout pig used the MC3R deletion clones as donor cells via somatic cell nuclear transfer and embryo transfer.In this study,we constructed MC3R knockout pig model quickly and efficiently by CRISPR/Cas9 system,that can lay the foundation for the research on physiological function and molecular mechanism of MC3R.