Chu Yuankui, Lü Changrong, Chen Dongmei, Cao Hui, Dou Zhongying
Shaanxi Branch of National Stem Cell Engineering & Technology Center, Laboratory of Molecular Biology for Agriculture, College of Animal Medicine, Northwest A & F University, Yangling 712100, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Apr;26(4):448-53.
In order to construct the recombinant retrovirus vector of human ngn3 gene and its packaging cell line, we successfully amplified the open reading frame (ORF) of ngn3 gene from human fetal pancreatic tissue by RT-PCR. The PCR products of human ngn3 gene was subcloned into pMD18-T vectors and sequenced. Results showed that its sequence was fully consistent with the ngn3 gene published in GenBank(GenBank Accession No. BC126468). The correct fragment was digested by EcoR I and Hpa I from recombinant pMD18-T vector and inserted into the same restriction enzyme sites of retroviral vector pMSCV-neo. We got recombinant retrovirus vector pMSCV-ngn3, which was identified by double restriction enzyme digestion and then transfected into PT67 cells by lipofectamine 2000. We established the PT67-ngn3 packaging cell line by G418 selection, which was detected by RT-PCR and immunohistochemistry staining. The detection results showed that the Ngn3 expressed at the mRNA and protein level in the packaging cell line. RT-PCR detection and electronic microscope analysis showed that the recombinant retroviral vector pMSCV-ngn3 was packaged into infectious virus particles and released into the supernatant of the cells. These results demonstrated that a PT67-ngn3 packaging cell line was successfully established, and this could facilitate the study of differentiation of the human fetal pancreatic progenitor cells into insulin-producing cells by using the ngn3 gene.
为构建人ngn3基因的重组逆转录病毒载体及其包装细胞系，我们通过RT-PCR从人胎儿胰腺组织中成功扩增出ngn3基因的开放阅读框（ORF）。将人ngn3基因的PCR产物亚克隆至pMD18-T载体并测序。结果显示其序列与GenBank中公布的ngn3基因（GenBank登录号：BC126468）完全一致。从重组pMD18-T载体上用EcoR I和Hpa I双酶切出正确片段，并插入逆转录病毒载体pMSCV-neo的相同酶切位点。我们得到重组逆转录病毒载体pMSCV-ngn3，经双酶切鉴定后，用脂质体2000转染PT67细胞。通过G418筛选建立了PT67-ngn3包装细胞系，并用RT-PCR和免疫组织化学染色进行检测。检测结果显示包装细胞系中Ngn3在mRNA和蛋白水平均有表达。RT-PCR检测和电子显微镜分析表明重组逆转录病毒载体pMSCV-ngn3被包装成有感染性的病毒颗粒并释放到细胞上清中。这些结果表明成功建立了PT67-ngn3包装细胞系，这有助于利用ngn3基因研究人胎儿胰腺祖细胞向胰岛素分泌细胞的分化。
