title: [Evaluation and application of exogenous gene expression system based on retroviral vector].
abstract: Currently, exogenous gene expression system based on retroviral vector has been widely used as efficient gene expression system in both gene therapeutic research and RNA interference. In this study, we evaluated the efficiency of exogenous gene expression mediated by the retroviral vector in mammalian cells. First, we constructed EGFP (enhanced green fluorescent protein) vector using pcDNA3.1(+) and retroviral vector pQCXIN as backbone vector respectively. Then, we transfected or infected HEK293 cells and CHO-K1 cells with above vector or corresponding retroviral virus, and measured the relative fluorescence intensity (RFI) of EGFP. The results showed that the RFI of the retroviral virus-infected cells was two times higher than that of the plasmid-transfected cells. Further experiments revealed repeated virus infection enhanced the expression of EGFP markedly, with RFI increasing twice after four rounds of virus infection. Furthermore, the EGFP expression in HEK293 cells mediated by the retroviral vector was more stable than transfected with plasmid pcDNA3.1(+). Finally, we further validated the efficiency of exogenous gene expression system based on the retroviral vector by expressing recombinant human activated protein C (rhAPC) in HEK293 cells. We obtained HEK293 cell lines with rhAPC expression between 10 and 15 microg/(10(6) cells d). In conclusion, the exogenous gene expression system based on the retroviral vector is an alternative method for the generation of stable and high-expressing mammalian cell lines.
authors.affiliation: Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Lingling
authors.initials: L
authors.lastname: Ye
authors.forename: Jian
authors.initials: J
authors.lastname: Xu
authors.forename: Shichong
authors.initials: S
authors.lastname: Li
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.forename: Xingmao
authors.initials: X
authors.lastname: Liu
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Wang
authors.forename: Zhaolie
authors.initials: Z
authors.lastname: Chen
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D011486:Protein C / Q000096:biosynthesis / Q000235:genetics; D034622:RNA Interference; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D011486:Protein C; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins