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Improvement in the suspension‐culture production of recombinant adeno‐associated virus–LacZ in HEK‐293 cells using polyethyleneimine–DNA complexes in combination with hypothermic treatment
Author(s) -
Feng Lei,
Guo Meijin,
Zhang Shuxiang,
Chu Ju,
Zhuang Yingping,
Zhang Siliang
Publication year - 2008
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20070081
Subject(s) - transfection , hek 293 cells , microbiology and biotechnology , adeno associated virus , recombinant dna , shuttle vector , genetic enhancement , cell culture , gene delivery , biology , plasmid , reporter gene , dna , chemistry , gene , biochemistry , vector (molecular biology) , gene expression , genetics
rAAV (recombinant adeno‐associated virus) has become a very useful gene‐delivery vector for gene therapy. However, it is very difficult to generate rAAV using triple transfection on a commercial scale, owing to its low transfection efficiency. An optimal procedure for transfection in suspension‐culture mode was developed for rAAV–LacZ production in suspension‐cultured HEK‐293 (human embryonic kidney‐293) cells mediated by PEI (polyethyleneimine)–DNA complexes in combination with transient severe hypothermia at 4 °C for 1 h in the present study (LacZ is the product of the reporter gene lacZ , which codes for β‐ d ‐galactosidase). It showed that the PEI/DNA ratio, cell density at the beginning of transfection and cell‐cycle arrest in G2/M‐phase were key factors affecting suspension‐culture triple‐transfection efficiency and rAAV–LacZ productivity. After incubation at 4 °C for 1 h and re‐warming at 37 °C for 18 h, HEK‐293 cells at 1×10 6 cells/ml were transfected with PEI–DNA complexes at a PEI/DNA ratio of 5:1 (w/w) with final concentrations of 30 μg/ml 25 kDa linear PEI and 6 μg/ml plasmid DNA in culture. After 6 h incubation for transfection, an equal volume of medium was added to the culture for additional 48 h growth until harvest. Finally, the high transfection efficiency of some 75% and rAAV–LacZ titre of (7.48±0.59)×10 11 physical particles or 1.86±0.96×10 10 infectious particles were achieved in 250 ml shake flasks with 60 ml working volume, indicating a promising application for scale‐up.