The Construction Of Several Skin-Specific Expression Vectors And The Indentification Of Transgenic Cells

Cashmere growth is a very complex process which is affected by the environmental and genetic factors, and it is closely related to the differentiation, formation of the hair follicle and the hair follicle cycle. In this process, it is regulated by some hair follicle growth inhibition genes such as FGF5, FGF18, and some genes that promote hair follicle growth such as EGF and VEGF etc. In the field of goat breed, it has become one of the research hot spots that used biotechnology import exogenous gene to the cells or knocked out endogenous gene so that the wool production was improved.The research includes two parts:one part, we built the skin specific Cre recombinase gene vector, that specifically knockout the gene inhibited the growth of hair follice in the future; another part is general expression vector including multiple cloning site that used to express specific functional genes was built, the research provides conditions to build the skin specific gene expression vector. In this experiment, we first cloned specific skin epidermis keratin expression for5promoter (Hum keratin5promoter, K5) and keratin14promoter (Hum keratin14promoter, K14) and Cre recombinase gene by PCR; and then through the reorganization by ligase we constructed three kinds of skin specificity of expression vectors, marked as pIRES2EGFP-KAP6Cre, pIRES2EGFP-K5and pIRES2-EGFP-K14, which based on pIRES2-EGFP-KAP6vector. Then pIRES2-EGFP-KAP6-Cre and pIRES2-EGFP-Cre were transfered into Inner Mongolia goat fetal fibroblast cells and skin fibroblast cells by lipofectamineTM2000, to obtain stable transfected cell clones and screened to obtain stable transfected cell clones. The integration of exogenous gene was detected by RT-PCR.The Results showed that we built pIRES2-EGFP-KAP6-Cre, pIRES2-EGFP-K5and pIRES2-EGFP-K14skin contains specific expression vector and pIRES2EGFP-Cre contained CMV promoter which was a nonspecific vector, which sequence and all components connected are correct by sequenced. We used pIRES2-EGFP-KAP6-Cre, pIRES2-EGFP-Cre vector to transfect in skin fibroblasts and fetal fibroblasts of the Inner Mongolia Cashmere goat. PCR results showed that exogenous gene had integrated into the cellular genome. The expression of pIRES2-EGFP-KAP6-Cre in skin fibroblasts was far higher than the expression in fetal fibroblasts, the expression of pIRES2-EGFP-Cre in two kinds of cells has no significant difference by Semi RT-PCR.This study successfully constructed four kinds of eukaryotic expression vector. Exogenous Cre recombinase gene was specific expressed in skin fibroblasts. By selecting those which could stable express green fluorescent protein, Cre recombinase specificity in skin fibroblasts of the Inner Mongolia Cashmere goat, we could provide nuclear donor for transgenic cloning technology.