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目的:对猪primed胚胎干细胞、囊胚内细胞团(inner cell mass,ICM)和胎儿成纤维细胞(porcine embryonic fibroblasts,PEF)转录组比较分析,在筛选出ICM中表达水平上调的5个转录因子(OCT4、TBX3、REX1、LIN28及DPPA5)的前期研究基础上,尝试构建具有2A肽(2A peptide)基因序列的转录因子重组表达载体,并与pEF1a-Tet3G质粒共转染PEF细胞,以期获得同时转入5个转录因子的单克隆细胞系,为讨论利用Tet-On 3G诱导表达系统并通过添加盐酸多西环素(doxycycline hyclate,DOX)激活转录因子表达,高效诱导形成猪诱导多能干细胞(induced pluripotent stem cells,iPS)的相关研究奠定基础.方法:以PEF细胞cDNA为模板,利用PCR方法扩增获得猪源REX1、LIN28、DPPA5 3个转录因子,并将3个转录因子以E2A和T2A序列连接成三因子片段(RLD),最终将三因子片段及商业合成的OCT4和TBX3连接到改造后的诱导表达载体pTRE3G-Zs中,获得3个重组表达载体;利用核转染的方法,将3个重组诱导表达载体与表达反式激活蛋白的pEF1a-Tet3G质粒共转染PEF细胞,并通过药物筛选和鉴定获得单克隆转基因细胞系.结果:扩增获得带有2A肽序列的猪源转录本片段REX1 (975bp)、UN28 (727bp)和DPPA5(408bp),并获得2A肽序列连接成的三因子片段RLD,最终构建了3个表达载体(pTRE3G-Zs-OCT4、pTRE3G-Zs-TBX3、pTRE3G-Zs-RLD),且经酶切鉴定证明载体连接正确;3个表达载体与pEF1 a-Tet3G质粒核共转染PEF细胞后,经过药物筛选和外源基因鉴定,共获得同时转入五因子的单克隆细胞系70个,其中29个细胞系外源基因拷贝数较高.结论:成功建立了具有高拷贝数猪源OCT4、TBX3、REX1、LIN28、DPPA5 5个转录因子的单克隆转基因细胞系.“,”Objective:Based on the comparison of transcriptional profiles between porcine primed embryonic stem cells(pESC),porcine inner cell mass(ICM) and porcine embryonic fibroblasts(PEF) in our previous study,five transcriptional factors(OCT4,TBX3,REX1,LIN28 and DPPAS) were selected due to their expressions were significantly higher in ICM than pESC and PEF.To establish porcine induced pluripotent stem (iPS) cell line,three expression vectors with five porcine transcriptional factors connected via 2A peptide gene sequence were constructed.The induced expression vectors and pEF1a-Tet3G were cotransfected into PEF followed by supplement with doxycycline hyclate to obtain more efficient pluripotent stem cell induction strategy.Methods:Firstly,the cDNA sequences of transcription factors REX1,LIN28,DPPA5 were cloned from PEF through PCR and linked together with E2A and T2A sequence (RLD).The cDNA sequences of transcription factors OCT4 and TBX3 were synthesized.Secondly,OCT4,TBX3 and RLD were transfected respectively into the TET-ON induced expression plasmid(pTRE3G-Zs),followed by plasmid extraction and restriction endonuclease digestion to verify the correct construction of three recombinant vectors.Finally,the transgenic cell lines were obtained via nucleofection of these three vectors and pEF1a-Tet3G,and were screened with G418.Results:975bp REX1 sequence,727bp LIN28 sequence and 408bp DPPA5 sequence were obtained by PCR.The correct construction of three recombinant vectors(pTRE3G-Zs-OCT4,pTRE3G-Zs-TBX3,and pTRE3G-Zs-RLD) was verified by restriction endonuclease.A total of 70 cell lines including 29 cell lines with multicopy of foreign genes were established by drug screening and PCR.Conclusion:We have established transgenic cell lines with five porcine transcription factors (OCT4,TBX3,REX1,LIN28,and DPPA5) and pEF1a-Tet3G plasmid successfully.