南京医科大学学报（自然科学版）2017，Vol.37Issue(11)：1377-1384,8.DOI:10.7655/NYDXBNS20171101
整合高拷贝数猪源转录因子的猪胎儿成纤维细胞系的建立
Establishment of transgenic porcine fetal fibroblast cell lines with multi-copy of porcine transcriptional factors
摘要
Abstract
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.关键词
猪转录因子/2A肽基因序列/诱导表达载体/转基因细胞系/iPS细胞Key words
porcine transcriptional factor/2A peptide gene sequence/induced expression vector/transgenic cell line/iPS cell分类
生物科学引用本文复制引用
张曼玲,陈袁,赵丽华,李艳如,金永,王俊政,姜海滨,陈俏羽,李荣凤..整合高拷贝数猪源转录因子的猪胎儿成纤维细胞系的建立[J].南京医科大学学报（自然科学版）,2017,37(11):1377-1384,8.基金项目
国家自然科学基金(31371487) （31371487）
