1] Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan. [2].
Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan.
Nat Protoc. 2014 Aug;9(8):1882-99. doi: 10.1038/nprot.2014.130. Epub 2014 Jul 10.
Baculovirus is a promising vector for transducing numerous types of mammalian cells. We have developed hybrid baculovirus vectors and protocols for the efficient transduction of a variety of cell lines, primary cells and stem cells, including bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ASCs). The hybrid vector enables intracellular minicircle formation and prolongs transgene expression. The advantages of this transduction protocol are that baculovirus supernatant alone needs to be added to cells growing in medium, and transduction occurs after only 4-6 h of incubation at room temperature (25 °C) with gentle shaking. The entire procedure, from virus generation to transduction, can be completed within 4 weeks. Compared with other transduction procedures, this protocol is simple and can confer efficiencies >95% for many cell types. This protocol has potential applications in tissue regeneration, as transduced cells continue to express transgenes after implantation. For example, transduction of rabbit ASCs (rASCs) with growth factor-encoding hybrid baculovirus vectors, as described as an example application in this protocol, enables robust and sustained growth factor expression, stimulates stem cell differentiation and augments tissue regeneration after implantation.
杆状病毒是一种很有前途的载体，可以转导多种类型的哺乳动物细胞。我们开发了杂交杆状病毒载体和方案，可有效地转导多种细胞系、原代细胞和干细胞，包括骨髓间充质干细胞（BMSCs）和脂肪来源的干细胞（ASCs）。杂交载体能够在细胞内形成微小环，并延长转基因的表达。该转导方案的优点是，只需将杆状病毒上清液添加到生长在培养基中的细胞中，在室温（25°C）下轻轻摇动孵育 4-6 小时后即可进行转导。从病毒生成到转导的整个过程可以在 4 周内完成。与其他转导程序相比，该方案简单，对于许多细胞类型的效率>95%。该方案在组织再生中有潜在的应用，因为转导的细胞在植入后会继续表达转基因。例如，本文描述的一个应用示例，即用生长因子编码的杂交杆状病毒载体转导兔脂肪来源的干细胞（rASCs），可实现强大且持续的生长因子表达，刺激干细胞分化，并增强植入后的组织再生。
