奶牛干细胞多能性基因Nanog和Lin28克隆分析及其逆转录病毒表达载体的构建与包装

Sequencing analysis of cow Nanog gene and Lin28 gene and construction of theirs recombinant retroviral expression vectors

以50-60日龄的Holstein奶牛胎儿原始生殖嵴组织为材料，通过RT—PCR的方法克隆奶牛Nanog与Lin28基因开放性阅读框（ORF）全序列。序列全长分别为903bp和618bp。所克隆的奶牛Nanog与Lin28基因序列与GenBank中已经发表的参考序列进行比对同源性均为100％。将扩增的基因片段经过酶切后亚克隆到反转录病毒载体pMSCVneo的多克隆位点，经过酶切、PCR和测序鉴定正确后，应用lipofectamine2000介导转染PT67包装细胞，经过14dG418连续抗性筛选，挑选抗性集落扩大培养。收集病毒上清经过RT—PCR与透射电镜检测，并用NIH3T3细胞测定病毒滴度。结果表明，病毒上清中含有逆转录病毒颗粒，Nanog与Lin28基因在筛选后的包装细胞PT67细胞中均有转录，病毒上清感染滴度值分别达到7．5×10^7 cfu／mL和9．19×10^7’cfu／mL。结果表明，本试验已经成功克隆出奶牛Nanog与Lin28基因开放性阅读框全序列，并建立高效、稳定产生包含有pMSCV-Nanog与pMSCV-Lin28质粒的感染性病毒颗粒的包装细胞株，为进一步产生奶牛诱导性多能干细胞（Induced pluripotent stem cells，iPSCs）奠定基础。

Two pairs of primers were designed and synthesized based on the mRNA sequences of bovine Nanog gene and Lin28 gene in the. GenBank,which were used to amplify the full open reading frames （ORF） sequences of bovine pluripotency genes Nanog and Lin28. Total RNA was extracted and purified using Trizol reagent from bovine primordial germ ridges of fetal cows at 50-60 days of gestation,and treated with DNase I to remove genomic DNA contamination. The ORF full sequences of bovine Nanog gene and Lin28 gene were amplified by RT-PCR technique, and subcloned into pMD18-T vectors to sequence. The ORF full length of cow Nanog gene is 903 bp,and Lin28 is 618 bp. The sequence homologies of cloned Nanog and Lin28 gene were 100% to the counterparts in the GenBank, respectively. Verified sequences of Nanog and Lin28 gene were inserted into retroviral vectors pMSCVneo to construct the recombinant retroviral expression vectors. The two recombinant retroviral plasmids were respectively transfected into PT67 packaging cells with lipofectamine2000 to produce infectious retroviral particles containing Nanog and Lin28 genes. About 14 days after G418 screening,the PT67 packaging cell lines were established,which could stably produce retroviruses with high efficiency. The viral titer was determined using NIH3T3 cells,and the titers of retroviral pMSCV-Nanog and pMSCV-Lin28 reached 7. 5 × 10^7cfu/mL and 9. 19 × 10^7cfu/mL, respectively. The transcriptions of Nanog and Lin28 mRNA in the established packing cell lines were verified by RT-PCR. The transfection efficiency of pMSCV-Nanog was 55.6% ,and pMSCV-Lin28 was 54.4% in PT67 cell lines. Our results showed bovine Nanog gene and Lin28 gene sequences were successfully amplified, the recombinant retroviral vectors containing bovine Nanog gene and Lin28 gene were constructed, and screened the stable virus-producing packaging cell lines with efficient expressing the genes of interest.