Optimization of transfection mediated by calcium phosphate for plasmid rAAV-LacZ (recombinant adeno-associated virus-beta-galactosidase reporter gene) production in suspension-cultured HEK-293 (human embryonic kidney 293) cells.
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 beta-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 microg/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 x 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.
Feng
Lei
L
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, People's Republic of China.
Guo
Meijin
M
Zhang
Shuxiang
S
Chu
Ju
J
Zhuang
Yingping
Y
Zhang
Siliang
S
D000256:Adenoviridae / Q000235:genetics*; D002118:Calcium / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D001616:beta-Galactosidase; D002118:Calcium