摘要
【目的】克隆徐淮山羊多能性转录因子0ct4、Sox2、K1f4和c—Myc的CDS片段,构建pMD19-T—oct4、pMD19-T—sox2、pMD19-T—k1f4和pMD19-T—c—myc重组质粒,随后构建含T7启动子的pcDNA3-oct4、pcDNA3-sox2、pcDNA3-k1f4和pcDNA3-c—myc重组质粒,通过体外转录,获得该4种多能性转录因子的mRNA,并使其在徐淮山羊成纤维细胞中稳定表达。【方法】应用RT—PCR方法分别从徐淮山羊睾丸组织、皮肤组织、小肠组织中扩增多能性转录因子Oct4、Sox2、K1f4和c—Myc基因编码全序列,将该4种基因分别克隆到pMD19-T载体,构建pMD19-T—oct4、pMD19-T—sox2、pMD19-T-k1f4和pMD19-T—c—myc重组质粒。然后将pcDNA3.0载体和pMD19-T重组载体双酶切后用T4连接酶连接,构建含有T7启动子的真核表达载体pcDNA3-oct4、pcDNA3-sox2、pcDNA3-k1f4和pcDNA3-c—myc重组质粒。各重组质粒分别用限制性内切酶XhoI、XbaI单酶切,酶切后质粒模版按照体外转录试剂盒说明体外转录获得各多能性转录因子的mRNA,并对获得的mRNA进行检测,确定其稳定性和浓度。按照脂质体(体积):mRNA(质量)为1:1的比例用脂质体2000转染。转染24 h后,利用Western blot技术、间接免疫荧光实验检测徐淮山羊多能性转录因子Oct4、Sox2、K1f4和c—Myc基因的mRNA在成纤维细胞中的表达。【结果】①克隆得到的徐淮山羊0ct4、Sox2、K1f4和c—Myc基因编码序列全长分别为1 083、962、1 434和1 320 bp,并经TA克隆测序验证,其CDS序列与绵羊、人、牛和猪等的序列相似性在89%以上;②体外转录获得的4种多能性转录因子的mRNA经脂质体转染徐淮山羊成纤维细胞,在成纤维细胞中均定位于细胞核;③4种多能性转录因子的mRNA在徐淮山羊成纤维细胞中表达的蛋白与预期大小一致,分别为38、34、50和48 kd。【结论】成功克隆了徐淮山羊Oct4、Sox2、K1f4和c—Myc基因,该4种多能性转录因子的mRNA能够在徐淮山羊成纤维细胞中稳定表达,为进一步研究Oct4、Sox2、K1f4和c—Myc基因的功能和山羊体细胞的重编程奠定基础。
【Objective】 The genes of Oct4, Sox2, Klf4 and c-Myc were cloned, the pMD19-T-oct4, pMD19-T-sox2, pMD19-T-klf4, and pMD19-T-c-myc recombinant plasmids were constructed, and then the pcDNA3-Oct4, pcDNA3 -Sox2, pcDNA3-Klf4 and pcDNA3-c-Myc recombinant plasmids containing T7 promoter were constructed. According to the in vitro transcription, the four mRNA transcription factors were obtained, and it was made to express stably in Xuhuai goat fibroblasts. 【Method】Through the testicular, skin and small intestine tissue, the Oct4, Sox2, Klf4, and c-Myc genes coding sequence were obtained by RT-PCR, and were cloned into pMD19-T vector, and then cloned into the eukaryotic expression pCDNA3.0 vector that contains T7 promoter, and then the pcDNA3-Oct4, pcDNA3-Sox2, pcDNA3-Klf4 and pcDNA3-c-Myc recombinant plasmids were constructed. Recombinant plasmids were linearized using XhoI and XbaI, in vitro transcription of the transcription factors was made to obtain the mRNA using the instructions of in vitro transcription, the mRNA was detected, its stability and concentration were determined, Lipofectamine 2000 transfection was carried out according to the proportion of Lipofectamine﹕mRNA=1﹕1. Western blot technique and immunofluorescence assay were used to detect the mRNA expression in Xuhuai goat fibroblasts 24 h post-transfection. 【Result】 The length of Xuhuai goat Oct4, Sox2, Klf4 and c-Myc genes coding sequence were 1 083 bp, 962 bp, 1 434 bp and 1 320 bp, and confirmed by TA cloning, the sequences similarity of coding sequences of sheep, human, cattle and pig were above 89%. The mRNA of four transcription factors by transfection to Xuhuai goat fibroblasts were localized in the nucleus. The mRNA of four transcription factors expression in fibroblasts proteins was consisted with the expected size, 38 kd, 34 kd, 50 kd and 48 kd. 【Conclusion】The Xuhuai goat Oct4, Sox2, Klf4 and c-Myc transcription factors were cloned successfully, and the four mRNA can express in fibroblasts stably, thus a foundation for further study of the function of the genes Oct4, Sox2, Klf4, c-Myc and goat somatic cell reprogramming is established.
出处
《中国农业科学》
CAS
CSCD
北大核心
2014年第7期1417-1426,共10页
Scientia Agricultura Sinica
基金
科技部转基因生物新品种培育重大专项(2011ZX08008-003)
国家高技术研究发展计划(2011AA100307-04)
江苏高校优势学科建设工程资助项目
