PMID- 17880281 OWN - NLM STAT- MEDLINE DCOM- 20080610 LR - 20081121 IS - 1470-8744 (Electronic) IS - 0885-4513 (Linking) VI - 50 IP - Pt 2 DP - 2008 Jun TI - 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. PG - 121-32 AB - 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 degrees C for 1 h in the present study (LacZ is the product of the reporter gene lacZ, which codes for beta-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 degrees C for 1 h and re-warming at 37 degrees C for 18 h, HEK-293 cells at 1x10(6) cells/ml were transfected with PEI-DNA complexes at a PEI/DNA ratio of 5:1 (w/w) with final concentrations of 30 mug/ml 25 kDa linear PEI and 6 mug/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)x10(11) physical particles or 1.86+/-0.96x10(10) infectious particles were achieved in 250 ml shake flasks with 60 ml working volume, indicating a promising application for scale-up. FAU - Feng, Lei AU - Feng L AD - State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, People's Republic of China. FAU - Guo, Meijin AU - Guo M FAU - Zhang, Shuxiang AU - Zhang S FAU - Chu, Ju AU - Chu J FAU - Zhuang, Yingping AU - Zhuang Y FAU - Zhang, Siliang AU - Zhang S LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - United States TA - Biotechnol Appl Biochem JT - Biotechnology and applied biochemistry JID - 8609465 RN - 0 (Drug Carriers) RN - 0 (Recombinant Proteins) RN - 9002-98-6 (Polyethyleneimine) RN - 9007-49-2 (DNA) RN - EC 3.2.1.23 (beta-Galactosidase) SB - IM MH - Adenoviridae/genetics/*metabolism MH - Cell Culture Techniques/methods MH - Cell Line MH - DNA/*administration & dosage/chemistry/*genetics MH - Drug Carriers/*chemistry MH - Hot Temperature MH - Humans MH - Kidney/*physiology MH - Polyethyleneimine/*chemistry MH - Protein Engineering/*methods MH - Recombinant Proteins/metabolism MH - Transfection/*methods MH - beta-Galactosidase/genetics/*metabolism EDAT- 2007/09/21 09:00 MHDA- 2008/06/11 09:00 CRDT- 2007/09/21 09:00 PHST- 2007/09/21 09:00 [pubmed] PHST- 2008/06/11 09:00 [medline] PHST- 2007/09/21 09:00 [entrez] AID - BA20070081 [pii] AID - 10.1042/BA20070081 [doi] PST - ppublish SO - Biotechnol Appl Biochem. 2008 Jun;50(Pt 2):121-32. doi: 10.1042/BA20070081.