Research Group Pharmaceutical Biotechnology, Faculty of Applied Natural Sciences, STEPs Institute, TH Köln - University of Applied Sciences, Chempark Leverkusen E28, Kaiser-Wilhelm-Allee, 51368 Leverkusen, Germany; Research Group Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
Research Group Pharmaceutical Biotechnology, Faculty of Applied Natural Sciences, STEPs Institute, TH Köln - University of Applied Sciences, Chempark Leverkusen E28, Kaiser-Wilhelm-Allee, 51368 Leverkusen, Germany.
Virology. 2019 May;531:40-47. doi: 10.1016/j.virol.2019.02.014. Epub 2019 Feb 23.
Viral vector particles derived from murine leukemia virus (MLV) mediate highly efficient stable gene transfer used in gene therapeutic approaches and in the generation of transgenic cell lines. However, the establishment of stable viral packaging cells (VPCs) is a time-consuming challenge. To overcome this limitation, we successfully generated novel Sleeping Beauty-derived transposon vectors entailing envelope and packaging expression cassettes as well as a transfer vector. Upon multiplexed transposition in human cells, VPC bulk populations yielding titers of over 1 × 10 transduction-competent vectors were established within three weeks. In contrast, conventional plasmid-based establishment of VPCs, conducted in parallel, took much longer and yielded significantly lower vector productivity and vector fitness. The generated MLV vectors decorated with the envelope proteins of ecotropic MLV PVC-211mc mediated efficient transduction of Chinese hamster ovary (CHO) cells. Cell susceptibility was further elevated upon recombinant expression of the murine ecotropic receptor mCAT employing a transposon vector.
源自鼠白血病病毒 (MLV) 的病毒载体颗粒介导高效稳定的基因转移，用于基因治疗方法和转基因细胞系的产生。然而，建立稳定的病毒包装细胞 (VPC) 是一项耗时的挑战。为了克服这一限制，我们成功地生成了新型的睡眠美人衍生转座子载体，包含包膜和包装表达盒以及转移载体。在人细胞中进行多重转座后，在三周内建立了产生超过 1×10 转导效价的 VPC 批量群体。相比之下，平行进行的传统基于质粒的 VPC 建立需要更长的时间，并且产生的载体产率和载体适应性显著更低。用 ecotropic MLV PVC-211mc 的包膜蛋白装饰的生成的 MLV 载体有效地转导中国仓鼠卵巢 (CHO) 细胞。通过使用转座子载体重组表达鼠的 ecotropic 受体 mCAT，进一步提高了细胞的易感性。
