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| Expression of Nanog gene with the mediation of lentiviral vector in mouse ES cells |
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Abstract 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.
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Received: 22 October 2008
Published: 06 May 2009
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