Premium
Optimization of transfection mediated by calcium phosphate for plasmid rAAV‐LacZ (recombinant adeno‐associated virus–β‐galactosidase reporter gene) production in suspension‐cultured HEK‐293 (human embryonic kidney 293) cells
Author(s) -
Feng Lei,
Guo Meijin,
Zhang Shuxiang,
Chu Ju,
Zhuang Yingping,
Zhang Siliang
Publication year - 2007
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/ba20060143
Subject(s) - transfection , hek 293 cells , microbiology and biotechnology , reporter gene , cell culture , adeno associated virus , gene delivery , biology , plasmid , recombinant dna , gene , vector (molecular biology) , biochemistry , 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 productivity and inconveniently adhesive nature of its culture. An optimal suspension‐culture transfection procedure was developed for rAAV‐LacZ production in suspended HEK‐293 cells mediated by calcium phosphate ( lacZ , a reporter gene, codes for β‐galactosidase). The study showed that cytotoxicity of transfection complexes and cell aggregation in suspension culture were two key factors affecting high suspension‐culture transfection efficiency. Cytotoxicity of transfection complexes was influenced effectively by mixture of Ca 2+ and plasmid DNA when their concentrations were decreased from 300 to 150 mM and from 3.0 to 1.5 μg/ml respectively, as manifested by a relatively higher cell viability after suspension‐culture transfection. Moreover, the transfection efficiency was still less than 15%. In addition, we explored the disruption of cell aggregation and the control of transfection‐complex size with 2.0 mM EGTA treatment for 30 min before transfection and the addition of 100 mM Mg 2+ during transfection respectively, procedures which enhanced transfection efficiency significantly, owing to more contact and endocytosis between cells and transfection complexes. Finally, the high transfection level and rAAV‐LacZ titre achieved under optimized suspension‐culture transfection conditions, namely 40% and 5×10 11 v.g. (vector genomes)/60 ml of medium respectively, is promising for the technique's application in the large‐scale production of rAAV.