| Electronic Journal of Biotechnology ISSN: 0717-3458 |
Vol.
13 No. 5, Issue of September 15, 2010 |
| © 2010 by Pontificia Universidad Católica
de Valparaíso -- Chile |
Received March 6, 2010
/ Accepted May 26, 2010 |
| DOI: 10.2225/vol13-issue5-fulltext-6 |
|
Optimization of
polyethylenimine-mediated transient transfection using response surface
methodology design
Qiangyi Fang*
Shanghai Institutes for
Biological Sciences
Chinese Academy of
Sciences
Shanghai, China
E-mail: hicell@gmail.com
Bingqian Shen
College of
Pharmaceutical Sciences
Nankai University
Tianjin, China
*Corresponding author
Keywords: bioreactor, human tissue kallikrein, transient
gene expression.
Abbreviations: |
ANOVA: Analysis of
variance
CHO: chinese
hamster ovary
GFP: green
fluorescent protein
HEK 293-F:
human embryonic kidney cell
hTK: human
tissue kallikrein
IOD: integral
optic density
PCR:
polymerase chain reaction
PEI:
polyethylenimine
RSM: response
surface methodology
TGE:
transient gene expression
TproK: tissue
prokallikrein |
Response surface methodology was undertaken to optimize the
polyethylenimine-mediated transient transfection of suspension cultured HEK
293-F cells. A total of 15 combinations were designed according to Box-Behnken
design to identify the effects of DNA concentration, polyethylenimine
concentration and incubation time on transient transfection efficiency. The
highest integral optic density of green fluorescent protein presenting
r-protein yield was accessed using a DNA concentration of 1.75 µg/mL, a
polyethylenimine concentration of 10.5 µg/mL, and an incubation time of 11.8
min. Analysis of variance demonstrated that the experimental values fit well
with a quadratic model. The RSM-optimized transient transfection resulted in
greater production of human tissue prokallikrein (TproK) than non-RSM-optimized
conditions: protein yield was 32.0 mg/L and the maximum viable cell density
reached 3.57 x 106 cells/mL in a 5 L
stirred-tank bioreactor culture.
|
