The Construction Of Skin-Cell-Specific Expression Vector Of LEF1 Of Inner Mongolia Cashmere Goat And Its Stably Transgenic Cell Lines

Lymphoid enhancer factor 1 (LEF-1) is one member of the family of LEF/T-Cell Factor, and core factor of classic Wnt Pathway. It serves regulating cellular differentiation and tissue renew, especially the development of fair follicle, through forming(3-catenin/LEF complex, binding withβ-catenin.Cloning the Lymphoid enhancer factor, LEF1 gene from Inner Mongolia Cashmere Goat, then constructed skin-cell-specific expression vectors pCDsRed-KL and pCDsRed-UL. Vectors were transferred into goat fetal fibroblasts Cells (GFb) and skin fibroblasts to obtain transgenic cell line, which stable expresses red fluorescence. The transgenic cell lines can be used to nuclear transfer.LEF1 gene was cloned by RT-PCR. Then pCDsRed-KL, a skin-cell-specific expression vector of LEF1, was constructed by connecting with KAP6.1 promoter, as well as a pCDsRed2 expression unit. The Inner Mongolia Cashmere Goat fetal fibroblast (GFb) cells were transfected with the expression vector by iipofectamineTM2000. Cell clones stably expressing red florescence was obtained after screening by G418. The recombinant of extrogenous DNA was identified by PCR. the promoter sequence of ultra high sulfur keratin (UHSP) of Mus mouse was cloned by PCR. Then, linked with LEF1 gene, UHSP was cloned into a eukaryotic expression vector pCDsRed2-1, constructing skin-cell-specific expression vector pCDsRed-UL. GFb cells were transfected with the expression vector by lipofectamineTM2000. Cell clones stably expressing red florescence was obtained after screening by G418. The recombinant of extrogenous DNA was identified by PCR. The primary cultured cells of Inner Mongolia Cashmere Goat skin tissue were obtained by explant tissue culture and skin fibroblasts were purified by enzymes digest, skin fibroblasts were transfected with pCDsRed-KL,pCDsRed-UL by lipofectamineTM2000 and screened by G418. Transcription of pCDsRed-KL,pCDsRed-UL was identified by RT-PCR.Experiment Result showed that:(1) The cloned LEF1 gene cDNA from Inner Mongolia Cashmere Goat was 1159bp in length, including an ORF of 1116bp (HM059927). The sequencing result showed the LEF1 gene was connected properly to the downstream of KAP6.1 promoter, then the CMV promoter and the DsRed2 gene in sequence. The stably transfected cells were selected by G418. Identification of the transgene in the cell clones was examined by PCR and the exogenous DNA has been integrated into genome. The stably transfected cell line can express the red fluorescence efficiently. (2) The cloned UHSP was 670bp in length. DNA sequencing shows that a linking of UHSP, LEF1 gene, CMV and DsRed on sequence and the construction of vector are correct. The stably transfected cells were screened by G418. Identification of the transgene in the cell clones was examined by PCR and the exogenous DNA has been integrated into genome. (3) skin fibroblasts were purified and transfected with pCDsRed-KL,pCDsRed-UL. The stably transfected cell line which expressed the red fluorescence efficiently was screened by G418. LEF1 can transcript in the skin fibroblasts transfected with pCDsRed-KL,pCDsRed-ULThe LEF1 gene was cloned, Skin-cell-specific expression vectors of LEF1 was constructed successfully. The transfection by lipofectamineTM 2000 was efficient. The exogenous genes have been integrated into GFb cells genome stably, and transcription of LEF1 gene was identified in the skin fibroblasts transfected with pCDsRed-KL,pCDsRed-UL. The transgenic cell line which expressed red florescence was obtained. Taken together, these data offers transgenic cell lines and can be used to obtain a transgenic Cashmere goat.