猪源Lin28基因的克隆及其在猪胎儿成纤维细胞中的表达  

Cloning of porcine Lin28 and over-expressing it in porcine fibroblasts

作　　者：刘忠华[1] 韩小娇[1] 张鑫淼[1] 王娟[1] 牟彦双[1] 何文腾[1] 黄天晴[1] 孔庆然[1] 姜丹丹[1] 丛义梅[1] 尹智[1] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030

出　　处：《东北农业大学学报》2012年第9期26-31,I0001,共7页Journal of Northeast Agricultural University

基　　金：国家"973"计划重大科学研究项目(2009CB941002;2009CB932004);黑龙江省杰出青年基金(JC200905)

摘　　要：Lin28广泛地表达于早期胚胎中,是重编程的一个重要诱导因子。根据GenBank中公布的猪源Lin28的序列设计合成引物,以猪桑葚胚cDNA为模板,通过RT-PCR方法扩增其编码区全序列。将此克隆片段连接到PMXs表达载体上,构建逆转录病毒载体PMXs-Lin28。在293T细胞中包装携带Lin28基因的逆转录病毒,感染猪如猪胎儿成纤维细胞(Porcine embryonic fibroblasts,PEF)。免疫荧光及Real-time结果显示,PMXs-Lin28载体能够介导Lin28 mRNA和蛋白质在猪胎儿成纤维细胞中的高效表达,Lin28基因能够激活Oct4、Sox2、Nanog、Klf4和c-Myc基因的表达。Lin28 is widely expressed in early embryos,and it is also an important reprogramming factor.Based on the sequence published in the Gene Bank,the coding region of porcine Lin28 gene was choned from porcine morula cDNA by RT-PCR.Then,the cloned fragment was inserted into the PMXs expression vector to construct the pMXs-lin28 retroviral vector.Retrovirus carring the porcine Lin28 gene was packaged in 293T cells using the pMXs-lin28 vector and was used to infect with porcine embryonic fibroblasts.The results of Real-time PCR and immunofluorescence displayed that the PMXs-Lin28 vector could mediate high Lin28 mRNA and protein expression level in porcine embyonic fibroblasts.The results of real-time PCR also showed that Lin28 could activate Oct4,Sox2,Nanog,Klf4,and C-myc.

关 键 词：猪 Lin28 成纤维细胞 过表达 

分 类 号：TM344.1[电气工程—电机]