Construction Of Cashmere Goat VEGF164Gene Eukaryotic Expression Vector And Transfection Cells By Using K14Promoter And CRE/LOXP System

Mongolian White Cashmere Goats is good goat breeds in China, its fine cashmere diameter and good comprehensive quality, which has the value of very high application.Transgenic cloning technology makes the exogenous gene over-expression in skin cells, and thus promote the follicular development and fluffy villi growth. There is a kind of effective way to foster high yield animal strains.Considering the safety of transgenic animals, selective to delete marker genes is the key to solve the security problems.This study through the using of K14promoter and Cre/LoxP knockout system, build VEGF gene eukaryotic expression vector of the skin specific expression.And this vector is named pKV-L,respectively stable transfection the vector into goat fetal fibroblasts cell and goat skin fibroblasts cell.After we got the stable expression of red fluorescent protein transgenic cell cloning, detection VEGF gene expression in cells in different. Screening transgenic cells, for further provide nuclear donor.Experiment was based on the vector pDsRed2by PCR technology in pDsRed2polyclonal site to add to loxP sequence and enzyme site,connection with K14VEGF-PolyA fragment, finally to add CMV promoter. Successful construction VEGF164genes specifically express in skin and selectively delete marker gene eukaryotic expression vector pKV-L.Inner Mongolia white goat fetal fibroblast cells and skin fibroblast cells are transfected by vector pKV-L by lipofectamineTM2000mediated.The amount of VEGF mRNA in goat fetal fibroblast and skin fibroblast cells was detected Semi-quantitative.Sequencing results show that the successful construction VEGF164genes specifically express in skin and selectively delete marker gene eukaryotic expression vector pKV-L.Vector component:K14promoter, VEGF164gene, K14PolyA, loxP, CMV promoter and red fluorescent protein are properly connected in sequence. By PCR detection pKV-L stable transfect cells and the exogenous gene integrate into the cellular genome.Semi-quantitative PCR showed that exogenous VEGF164had expressed in cell.Skin fibroblasts is significantly higher than the amount of expression in fetal fibroblasts.This study successfully build eukaryotic expression vector of stable red fluorescent protein expression and VEGF164genes specifically express in skin.both to achieve the specific expression, and realized the purpose can be conditioned to delete marker genes. Using People K14promoter in goat cells with skin characteristics of the specific expression. In order to further get new strains of hair follicle by transgenic cloning technology specific gene over-expression VEGF164goat.We got the transgenic cells for the establishment of the hair follicle specificity over-expression lines to provide the basis. Cre/LoxP knockout system was introducted Successly,as the progeny of transgenic products introduced in exogenous delete laid the foundation of the resistance genes, genetic screening, to ensure the safety of genetically modified products.