ABSTRACT
Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre-based cell engineering. Biotechnol. Bioeng. 2016;113: 1600-1610. © 2016 Wiley Periodicals, Inc.
Subject(s)
Gene Targeting/methods , Genetic Vectors/genetics , Integrases/genetics , Retroviridae/genetics , Transgenes/genetics , Animals , CHO Cells , Cricetinae , CricetulusABSTRACT
Purpose:To investigate the effect of replication defective retroviral vectors carried sense or antisense TGF?1 fragment on the cell cycle regulation and proliferation of human bladder cancer. Methods:The replication defective retroviral vectors that integrated sense or antisense bioactive fragment of transforming growth factor?1 were constructed,and named as pRevT? and pRevT?-AS respectively. The influence of each vector on the cell proliferation,clone-formation and alteration of cell cycle of bladder cancer cell line EJ were observed in vitro.Results:The titre of pRevT? and pRevT?-AS were 0.84,0.88?10 5 CFU/ml respectively,the vectors integrated to EJ cells and expressed efficiently. Inhibition TGF?1 gene expression reduced proliferation and clone-formation rates of EJ cells. The G 0 /G 1 stage ratios in the antisense TGF?1-transfected EJ cells were increased,simultaneously,the S stage ratios were decreased. Conclusions:The antisense TGF?1 vector can reduce the expression of endogenous TGF?1 in EJ cells,induce G 1 stage arrest and inhibit proliferative growth in vitro.
ABSTRACT
In order to use retroviral-mediated gene transfer technology in clinical application, retroviral vector must be of high titer and free of detectable replication-competent retroviruses (RCR). The aim of this study was to optimize methods of defective retroviral vector production. Study was conducted using a LXSN vector inserted with human tumor necrosis factor-? gene and an amphotropic retrovirus packaging cell line-PA317. The results indicated that viral titer was influenced by volume of medium and concentration of fetal calf serum. Inactivation of retroviral vector was greater at 37癈 than at 32癈. In experiment of transfection of PA317 and transduction of 3T3, integration of retroviral vector into genome of packaging cells and target cells, and free of RCR were detected by polymerase chain reaction analysis. Viral vector with high titer and free of RCR is able to use in clinical trial
