Bryson Paul D, Zhang Chupei, Lee Chi-Lin, Wang Pin
Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles, CA, USA.
J Vis Exp. 2013 Jun 19(76):50606. doi: 10.3791/50606.
Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.
慢病毒载体（LVs）是将遗传物质传递到多种类型细胞的有力手段。由于与这些源自HIV-1的载体相关的安全问题，大量生产慢病毒载体具有挑战性。在本文中，我们报告了一种生产高滴度自失活慢病毒载体的方法。我们通过逆转录病毒转导四环素调控稳定生产细胞系GPR，以产生一个能够表达所有病毒成分的细胞系GPRS，这些成分包括一种树突状细胞特异性糖蛋白SVGmu。然后，我们使用串联DNA转染法，将编码报告基因绿色荧光蛋白（GFP）并结合一个选择标记的慢病毒载体转移质粒进行转染。所获得的几个克隆产生慢病毒载体的滴度比我们通过瞬时转染所达到的滴度高10倍。此外，这些病毒在体外能有效地转导树突状细胞，并对我们的报告抗原产生强烈的T细胞免疫反应。这种方法可能是生产用于癌症或传染病临床研究的强效慢病毒载体疫苗的一个不错选择。
