摘要
【目的】克隆内蒙古白绒山羊胸腺素β4(thymosinbeta4,Tβ4)基因,构建皮肤特异性表达载体,转染内蒙古白绒山羊胎儿成纤维细胞,筛选出稳定表达红色荧光蛋白并可用于核移植的转基因细胞克隆。【方法】通过RT-PCR克隆Tβ4基因cDNA序列,然后与KAP6-1基因启动子片段以及红色荧光蛋白表达元件连接构成Tβ皮肤特异性表达载体pCDsRed-KT。外源表达载体以lipofectamineTM2000介导转染胎儿成纤维细胞,通过G418筛选获得稳定转染的细胞克隆。PCR鉴定外源基因在细胞基因组中的整合。【结果】克隆了内蒙古白绒山羊Tβ4基因,cDNA全长142bp,其中包含135bp的完整ORF,编码44个氨基酸残基,氨基酸序列与已报道的牛胸腺素β4(NM_001002885)同源性为100%。测序显示构建的表达载体pCDsRed-KT中,Tβ4基因正确连接在皮肤特异性启动子KAP6-1下游,顺序连接CMV启动子和红色荧光蛋白基因,载体构建正确。PCR检测显示外源KAP6-1启动子和Tβ4基因整合到细胞基因组中,筛选出的转基因细胞高效表达红色荧光蛋白。【结论】克隆得到内蒙古白绒山羊Tβ4基因并构建成功其真核表达载体,可稳定转染绒山羊胎儿成纤维细胞,为下一步通过核移植方法获得转胸腺素β4基因绒山羊提供了条件。
【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 （NM_001002885）. The sequencing result showed that the Tβ4 gene was connected properly to the downstream of pKAP6-1, 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.
出处
《中国农业科学》
CAS
CSCD
北大核心
2010年第21期4497-4504,共8页
Scientia Agricultura Sinica
基金
国家自然科学基金(30860191)
国家基础科学人才培养基金(J0730648)
转基因生物新品种培育重大专项课题研究计划(2008ZX08008-002)
关键词
内蒙古白绒山羊
胸腺素Β4
皮肤特异性表达载体
稳定转染
inner Mongolia Cashmere Goat fetal fibroblast
Thymosin beta 4（Tβ4）
skin-follicle-cell-specific expression vector
stable transfection
