Sena-Esteves Miguel, Tebbets Jessica C, Steffens Sabine, Crombleholme Timothy, Flake Alan W
Department of Surgery, The Children's Hospital of Philadelphia, Abramson Research Center, 3615 Civic Center Blvd., Philadelphia, PA 19104, USA.
J Virol Methods. 2004 Dec 15;122(2):131-9. doi: 10.1016/j.jviromet.2004.08.017.
The goal of the present study was to develop an efficient transient transfection method for large-scale production of high titer lentivirus vector stocks of eight different pseudotypes. The envelope genes used for this purpose were those from VSV-G, Mokola, Rabies, MLV-Ampho, MLV-10A1, LCMV-WE, and LCMV-Arm53b. All envelopes were cloned into phCMV, which yielded lentivirus vector titers one, two, or three orders of magnitude higher than the original plasmids for the Rabies, MLV-10A1, and MLV-Ampho envelopes, respectively. When these newly constructed envelope expression plasmids were used for packaging, treatment with sodium butyrate resulted in almost five-fold increase in titers for some of the pseudotypes, had no effect for others (VSV-G and Rabies), and negatively impacted titers for the LCMV-derived pseudotypes. Production of vectors in serum-free media yielded titers only slightly lower than those obtained in the presence of serum. The efficiency of concentrating vector supernatants by ultracentrifugation or ultrafiltration was compared, with higher recovery efficiencies for the latter method, but the highest titers for most pseudotypes were obtained by ultracentrifugation. The best conditions for each individual pseudotype yielded lentivirus vector stocks with titers above 1 x 10(9) tu/mL for most pseudotypes, and higher than 1 x 10(10) tu/mL for VSV-G.
本研究的目标是开发一种高效的瞬时转染方法，用于大规模生产八种不同假型的高滴度慢病毒载体储备液。用于此目的的包膜基因来自水疱性口炎病毒糖蛋白（VSV-G）、莫科拉病毒、狂犬病病毒、莫洛尼鼠白血病病毒（MLV）-嗜异性、MLV-10A1、淋巴细胞脉络丛脑膜炎病毒（LCMV）-WE株和LCMV-Arm53b株。所有包膜均克隆到phCMV中，这分别使狂犬病病毒、MLV-10A1和MLV-嗜异性包膜的慢病毒载体滴度比原始质粒高出一个、两个或三个数量级。当这些新构建的包膜表达质粒用于包装时，丁酸钠处理使某些假型的滴度几乎提高了五倍，对其他假型（VSV-G和狂犬病病毒）没有影响，而对LCMV衍生的假型滴度有负面影响。在无血清培养基中生产载体产生的滴度仅略低于在有血清存在时获得的滴度。比较了通过超速离心或超滤浓缩载体上清液的效率，后一种方法的回收效率更高，但大多数假型的最高滴度是通过超速离心获得的。每种单独假型的最佳条件产生的慢病毒载体储备液，大多数假型的滴度高于1×10⁹转导单位/毫升，VSV-G的滴度高于1×10¹⁰转导单位/毫升。
