慢病毒介导Nanog基因在小鼠ES细胞的表达

试验尝试构建小鼠Nanog基因慢病毒表达载体，培养表达外源Nanog基因的小鼠ES细胞。结果显示通过RT-PCR扩增出918bp的小鼠Nanog基因，测序正确的小鼠Nanog基因通过慢病毒介导在小鼠ES细胞表达后，表达外源Nanog基因的小鼠ES细胞生长状态同普通ES细胞无明显差异，在无LIF的ES细胞培养液培养条件下，表达外源Nanog基因的小鼠ES细胞保持正常的ES细胞集落，碱性磷酸酶、Oct4和SSEA-1免疫细胞化学检测为阳性，相同情况下未表达外源Nanog基因的小鼠ES细胞集落退化消失。试验证实了通过慢病毒载体介导培养了表达外源Nanog基因的小鼠ES细胞。试验尝试构建小鼠Nanog基因慢病毒表达载体，培养表达外源Nanog基因的小鼠ES细胞。根据小鼠Nanog基因m RNA序列设计Nanog基因引物，引物两端带有Nhe I和Xho I酶切位点。Trizol试剂处理小鼠ES细胞，通过RT-PCR扩增出小鼠Nanog基因，小鼠Nanog基因用Nhe I和Xho I酶切后连入pcDNA3.1载体中，PCR检测阳性的细菌克隆进行测序，测序正确的Nanog基因片段连接入PLL-IRES-Neo慢病毒表达载体中，包装含有Nanog基因的慢病毒感染小鼠ES细胞，在SNL细胞饲养层上G418筛选2周后，添加普通ES细胞培养液在普通小鼠胎儿成纤维细胞饲养层上培养。结果显示通过RT-PCR扩增出918 bp的小鼠Nanog基因，测序正确的小鼠Nanog基因通过慢病毒介导在小鼠ES细胞表达后，表达外源Nanog基因的小鼠ES细胞生长状态同普通ES细胞无明显差异，在无LIF的ES细胞培养液培养条件下，表达外源Nanog基因的小鼠ES细胞保持正常的ES细胞集落，碱性磷酸酶、Oct4和SSEA-1免疫细胞化学检测为阳性，相同情况下未表达外源Nanog基因的小鼠ES细胞集落退化消失。试验证实了通过慢病毒载体介导培养了表达外源Nanog基因的小鼠ES细胞。

Expression of Nanog Gene with the Mediation of Ientiviral Vector in Mouse ES Cells

In order to further study mouse embryonic stem cells(ES cells), lentiviral vector PLL-IRES-Nanog-Neo was constructed and mouse ES cells overexpressed nanog gene were cultured. At first, mouse nanog primers with Nhe I and Xho I restricted enzyme were designed according to nanog mRNA sequence. Nanog gene fragment was amplified by RT-PCR with mRNA as templates from ES cells treated with Trizol reagent. pcDNA3.1-Nanog vector were construted after nanog DNA fragments were digested with Nhe I and Xho I. Nanog fragment was confirm to original sequence by DNA sequence and then lentiviral vector PLL-IRES-Nanog-Neo was constructed. Mouse ES cells overexpressed nanog by mediation of lentiviral were cultured on mouse fetal fibroblast feeders after 2 weeks under G418 media and examined according to gowth characteristics. Results were showed that 918 bp nanog fragments were expressed in mouse ES cells mediated by lentiviral vector PLL-IRES-Nanog-Neo, mouse nanog- ES cells were taken on mass-like image and positve with alkaline phosphatase staining and Oct4 and SSEA1 immunocytochemistry under no LIF condition in the media. It is concluded that mouse ES cells Elevated nanog gene expression by mediation of lentiviral were constucted and cultured.