[Improving baculovirus transduction of mammalian cells by spinoculation].
Recently many reports have described that recombinant baculovirus could serve as a new gene transfer vehicle for mammalian cells with many unique advantages. In this study, the constructed recombinant baculovirus BacV-CMV-EGFPA containing the enhanced green fluorescent protein (eGFP) gene driven by CMV promoter was used to explore the feasibility of improving the efficiencies of transduction experiment in CV-1 cells by centrifugal method. Refer to the centrifugal transduction protocol of recombinant lentivirus, CV-1 cells were incubated with the culture supematant of Sf-21 cells infected by BacV-CMV-ECFPA (moi = 30) and then centrifuged at 600g for 1 h at RT, reporter gene transfer and expression efficiencies were analyzed by flow cytometry (FCM) 48h post transduction. Results showed that centrifugal method can achieve higher gene delivery and expression efficiencies than transduction by simple virus-cell mixing for 4h at 27 t with least impairment to cell viability. The centrifugal transduction protocol was further optimized by testing different centrifugal times, post-centrifugation incubation times and surrounding solutions. We found that centrifugation at 600g for 1 h at RT is sufficient to achieve the highest transduction efficiencies in target cells and PBS is more suitable than other surrounding solutions. Compared with previous protocol in which tranduction occurs for 4 - 8h at 27 degrees C, centrifugal method developed in this study could achieve more higher transduction efficiencies in more shorter time. Nine different mammalian cell lines (CV-1, 293FT, HepG2, 293T, CHO, C127, MT4, H9, Molt-4) were used to investigate the feasibility of delivering exogenous genes into different mammalian cells with the BacV-CMV-EGFPA supernatant (moi = 30) at 600g for 1h in PBS surrounding solution at RT. Results showed that most mammalian cell lines used in this study could be effectively transduced with recombinant baculoviruses by centrifugal method, and more higher and satisfactory transduction efficiencies could be achieved in primate adherent culture cells than in suspended culture cells. These results show that the baculovirus centrifugal transduction protocol have notable advantages: more rapid, efficient and nontoxic, and could be easily used in daily common experiments.
Cheng
Tong
T
National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, Xiamen University, Xiamen 361005, China.
Yang
Bing-Chun
BC
Xu
Chen-Yu
CY
Zhang
Tao
T
Du
Hai-Lian
HL
Wang
Ying-Bin
YB
Zhang
Jun
J
Xia
Ning-Shao
NS
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002498:Centrifugation / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D005240:Feasibility Studies; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D018411:Spodoptera; D014161:Transduction, Genetic / Q000379:methods*
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins