Construction And Identification Of An Epidermal Tissue Specific Expression Vector For HIF-1α Gene By Using CRE/LOXP System

Hair is a skin appendage which is derived from the skin during the evolution process. In generally, mammal hair growing goes througth a process of hair follicle’s development and differenciation.Many functional genes, growth factors are involed in this course. By transgenic technologies, we could successfully introduce a foreign gene into mammars cells, which can promote the hair growth, and obtain high yield and quality transgenic animals. At the same time, we have to solve the problem of biosecurity of these genetically modified products. The key point of this is how to remove the unexpected effects which is caused by the exogenous marker genes. In this study, by the specific knocking out function of Cre-LoxP system,we built pKH-L vector which can delete exogenous marker gene conditionally. This improved the safety of the transgenic products. Meanwhile, it provides an expression vector for transgenic cashmere goats production.Firstly, HIF-la gene was amplified by polymerase chain reaction according to the sequence of cDNA of Inner mongolian cashmere goat. Secondly, after the sequencing, pKT-L vector was used as cloning backbone, and pMD19T as an intermediate vector, the pKH-L eucaryotic expression vector was constructed (the order of functional elements are as K14-HIF-1α-PolyA-LoxP-CDsRed-LoxP). By liposome-mediated transfection, Inner Mongolian Cashmere Goats fetal fibroblasts and skin fibroblasts were transfected with the pKH-L vectors respectively. The fluorescence microscope observation showed that the transfection rate were both 65%. SYBR Green fluorescent real-time PCR detecting the mRNA level of HIF-1α gene in two kinds of cells results showed that it was significantly increased in skin fibroblasts group. Finally,we have detected the HIF-1α protein level in these two kinds of cells by western blot. The results displayed that it was also increased significantly in skin fibroblasts group. That means the construction process was success.In this study, we built skin-specific expression of HIF-1α gene targeting vector which could specific express in the Inner Mongolia Cashmere Goats skin fibroblast. It provides expression vector for transgenic goat, and ensures the safety of genetically modified products.