Based On Skin Cre/Loxp System Specific Expression Of Thymosin B4Gene Vector And Transgenic Cell Line Screening

Hair is the skin appendages, mammalian hair growth and differentiation of follicular development is relatesd to the regulation of genes involved in many functions and growth factors. According to transgenic technology, it has been a high-yield that scientists transfer exogenous genes into mammal cell, making skin-specific expression, promote hair growth. People benefit in the use of genetically modified products, but also must pay attention to the safety of genetically modified problem. In order to solve this problem, how to remove the marker gene is a key. According to this study, Cre-LoxP system was used to specific knockout feature, so we constructed a skin-specific expression vector of T(34gene marker gene, selective deletion, genetically modified to increase their security. And provided cashmere goats bred transgenic nuclear donor.First we built a middle expression vector pMD19T-K14-T(34-PolyA, further introducing Cre/Loxp knockout system by PCR techniques. In pDsRed2-l vector as the template, Cloning of Human CMV promoter, Construction of the skin condition-specific expression of thymosin (34of targeting vector pKT-L (functional element sequence K14-Tβ4-PolyA-LoxP-CDsRed-LoxP), By liposome-mediated transfection Inner Mongolia Cashmere Goats were fetal skin fibroblasts, after transfection in observed under a fluorescence microscope red fluorescence of cell confluence over50%, Adding an appropriate amount of G418selection, after a week, you can get stable expression of red fluorescent protein transgenic cell clones. Cell genome by PCR validation of exogenous genes to successful integration, testing Tβ4mRNA abundance in both cells using semi-quantitative RT-PCR method, Western blot were used to detect the expression level of these two cell genome Tβ4protein.Sequence analysis showed that we successfully constructed skin condition-specific expression Tβ4gene of the targeting vector pKT-L, and each component contained keratin14promoter, Tβ4gene, PolyA, CMV promoter, LoxP sequences and red fluorescent protein according to connecting the correct order. PCR was used to verify the situation within the cell genome integration of exogenous Tβ4gene. RT-PCR semi-quantitative experimental transit Tβ4cell genome gene significantly increased Tβ4mRNA expression, Western blot showed that after transfection Tβ4gene cells, the expression of the protein is also greatly increased, Tβ4mRNA expression levels in the skin higher amount of fiber cells than in fetal fibroblast cells, K14indicate the occurrence of skin-specific promoter role.In this study, we constructed the skin condition-specific expression of Tβ4targeting vector which could specific express in the Inner Mongolia Cashmere Goats skin fibroblast. And the pKT-L-transfected cashmere goat fetal fibroblast cells stability expressed red fluorescent protein. Those cells that tranfected Tβ4gene cloning were successful introduced of Cre/LoxP knockout system. Deleted exogenous marker genes for offspring genetically modified products introduced in the foundation, and provide the conditions for protecting the safety of genetically modified.