| Mammalian hair growth regulation is a very complex process which is experienced hair follicle development, differentiation and hair growth, and it is subject to a number of functional genes and other growth factors. Hair follicle-specific expression exogenous gene for cashmere growth, and cultivation of high-yielding cashmere rate new lines of transgenic animals was one of the hotspots in recent years. However, the security of transgenic animals was a very important problem in the cultivation of transgenic animals, how to delete the marker genes used to select the transgenic cell clones was the key to solve the security issues. In this study, selecting the hair follicle-specific promoter and constructing the conditional delete the marker gene vector were the most main content. Firstly, cloning of the human skin keratin14promoter (K14p), a hair follicle cell-specific promoter, then building pK14-DsRed2-1vector to test the specificity of K14p. In the construction of the conditional targeting vector, Cre/LoxP knockout system was used to build target vector pCDsRed2-K14VL which express goat VEGF164specificity in hair follicle, last transfect the vector into goat fetal fibroblast cells and skin fibroblast, respectively. It is selected that the clones which express the red fluorescent protein stably, and detected the quantity of VEGF164in different cells. Finally, we got the nuclear transfer cell donor, which specificly expresses exogenous VEGF164in the hair follicle and the marker genes can be deleted by the Cre enzyme.Human skin keratin14promoter was cloned by PCR, and used the web software analyzing its’promoter information. Keratin14promoter was subcloned into the upstream of the red fluorescent protein gene (DsRed) in pDsRed2-l vector to get the vector pK14-DsRed2-1, which could test the specificity of the promoter in hair follicles. Through the fusion PCR, LoxP sequences were inserting the target vector pCDsRed2-K14VL that expresses VEGF164in hair follicle specificity. Then conditional targeting vector pCDsRed2-K14VL was transfered into Inner Mongolia goat fetal fibroblast cells and skin fibroblast cells by lipofectamine TM2000, and G418screening to obtain stable transfected cell clones. The integration of exogenous gene was identified by PCR, and the quantity of mRNA in such two kinds of cells was detected by SYBR Green fluorescent real-time PCR, the quantity of VEGF164was detected by Western blot.Human epidermal keratin14promoter which cloned by the PCR was contained2274bp, and had99.5%identity with the K14p which reported in NCBI (DQ343282.1). The G+C%in nucleotide sequences was56%. The TATA box located at2169bp, and the CpG island sequences began at2061bp, end at2265bp. The conditional targeting vector pCDsRed2-K14VL which expresses VEGF164was built successfully. And the K14p, VEGF164, K14PolyA, CMV promoter, Loxp and red fluorescent protein gene are connected in the correct order. The screened transgenic cell clones were obtained, which Cashmere goat fetal fibroblasts and skin fibroblasts were transfected by pCDsRed2-K14VL. PCR detection showed that exogenous gene was integrated into the genome of cells. RT-PCR and western blot detection showed that the quantity of VEGF164mRNA and VEGF164proteins in transfected Inner Mongolia goat skin fibroblasts cells was much more than that in control Inner Mongolia goat fibroblasts cells. It showed that exogenous VEGF164gene was specific expressed in skin fibroblasts.In conclusion, K14promoter was cloned successfully and the hair follicle-specific function of K14promoter was verified successfully. Conditional target eukaryotic vector which express VEGF164specifically in hair follicles were built successfully. The VEGF164gene was specific expressed in skin fibroblasts of Inner Mongolia Cashmere goat. Cre/LoxP knockout system was introduced expression vector to delete the exogenous marker genes in progeny of transgenic animals. In this way, it can be achieved that the safety of genetically modified products. |
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