Abstract:

【Objective】 The objective of the experiment is to clone the thymosin beta 4 (Tβ4) gene from Inner Mongolia Cashmere Goat and then construct a skin-follicle-cell-specific expression vector pCDsRed-KT. The vector was transferred into goat fetal fibroblasts Cells (GFb) to obtain a transgenic cell line, which stably expresses red fluorescence. The transgenic cell line can be used for nuclear transplantation. 【Method】 Tβ4 gene was cloned by RT-PCR. Then pCDsRed-KT, a skin-follicle-cell-specific expression vector of Tβ4, 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 lipofectamineTM2 000. Cell clones stably expressing red florescence were obtained after screening by G418. The recombinant of extrogenous DNA was identified by polymerase chain reaction. 【Result】 The cloned Tβ4 gene cDNA from Inner Mongolia Cashmere Goat was 142bp in length, including an ORF of 135bp and encoding an active peptide which formed by 44 amino acids. The amino acid sequence shares 100% identity with the bovine Tβ4 (XM002706880.1). The sequencing result showed that the Tβ4 gene was connected properly to the downstream of pKAP6-, then the CMV promoter and the DsRed2 gene in sequence. Identification of the transgene in the cell clones was examined by PCR and the exogenous DNA (pKAP6-1 and Tβ4 gene) had been integrated into genome. The stably transfected cell line could express the red fluorescence efficiently. 【Conclusion】 The Tβ4 gene was cloned and a skin-follicle-cell-specific expression vector of Tβ4 was constructed successfully .The exogenous genes have been integrated into GFb cells genome stably. These results have paved the way to obtain the transgenic sheep by nuclear transfer in the future.

Key words: inner Mongolia Cashmere Goat fetal fibroblast')">inner Mongolia Cashmere Goat fetal fibroblast, Thymosin beta 4(Tβ4), skin-follicle-cell-specific expression vector, stable transfection