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Abstract According to the known mRNA sequence of bovine Oct4 gene in GenBank，a pairs of primers containing restriction sites of EcoR I and BglⅡ were designed, which were ultilized to amplify full sequence of ORF of bovine Oct4 gene. Total RNA was obtained from the primodial genital ridges of bovine fetus. The full-length of ORF of bovine Oct4 gene was 1,083 bp, which was obtained by RT-PCR reaction. The Homology of sequence cloned Oct4 gene was 99.4% to the counterpart sequence in Genbank. The recombinant plasmid pMSCV-Oct4 was transfected into packaging PT67 cells by lipofectamine 2000, at the same time, pMSCVneo-IRES-GFP vector (pMIG) was transfected into packaging PT67 cells as a control, which was used to measure transfected efficiency by flow cytometry analysis. Subsequently, we established a cell line stable producing retrovirus particles by G418 selection for 2 weeks. Retroviral titer was determinated with plaque formation method in NIH3T3 cell line, which was not less than 8.83×107cfu/mL. The transfection efficiency of pMSCV-Oct4 in PT67 cell line was 53.1%. The results indicated that the full sequence of bovine Oct4 gene was successfully cloned, and constructed recombinant retroviral pMSCV-Oct4 expression vector. Moreover, we established a packaging cell line stable producing retroviruses of Oct4 gene. The study must be helpful to the generation of induced pluripotent stem cells from bovine somatic cells.
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Received: 30 May 2009
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