A simple, rapid method for evaluation of transfection efficiency based on fluorescent dye.
Enhanced transfection efficiency of transient gene expression (TGE) and electroporation is a useful approach for improvement of recombinant therapeutic proteins in mammalian cells. A novel method is described here in which CHO cells expressing recombinant FVII (rFVII) were labeled with fluorescent dye and analyzed by confocal microscopy. Cells with or without rFVII encoding gene were detectable by flow cytometry. Thus, we were able to distinguish positive cells (with rFVII encoding gene) and quantify their percentages. We evaluated the effects of varying electroporation conditions (voltage, number of repetitions, plasmid amount, carrier DNA) in order to optimize transfection efficiency. The highest transfection efficiency achieved was ∼86%. The method described here allows rapid evaluation of transfection efficiency without co-expression of reporter genes. In combination with appropriate antibodies, the method can be extended to evaluation of transfection efficiency in cells expressing other recombinant proteins.
a The Key Laboratory of Industrial Biotechnology, Ministry of Education , School of Biotechnology, Jiangnan University , Wuxi , China.
Lin
L
Peng
b Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
Wendian
W
Xiong
b Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
Yanfei
Y
Cai
b Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
Yun
Y
Chen
c Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University , Suzhou , China.
Yang
Y
He
d Cyrus Tang Hematology Center and Ministry of Education Engineering Center of Hematological Disease , Soochow University , Suzhou , China.
Jianfeng
J
Yang
b Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
Jian
J
Jin
a The Key Laboratory of Industrial Biotechnology, Ministry of Education , School of Biotechnology, Jiangnan University , Wuxi , China.
Huazhong
H
Li
D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D018274:Electroporation / Q000379:methods; D005167:Factor VII / Q000235:genetics* / Q000378:metabolism*; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D014162:Transfection / Q000379:methods*
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D005167:Factor VII
CHO cells; electroporation; fluorescent dye; transfection efficiency
J Biol Chem. 2003 Feb 14;278(7):4826-30
12473656
Ann Anat. 2003 Jan;185(1):1-20
12597123
N Biotechnol. 2015 Dec 25;32(6):673-9
25529337
Biotechnol Prog. 2015 Nov-Dec;31(6):1685-92
26193826
Curr Opin Investig Drugs. 2006 Aug;7(8):750-8
16955687
Biotechnol Prog. 2014 Jan-Feb;30(1):132-41
24106171
Biotechnol Bioeng. 2008 Feb 15;99(3):578-87
17680677
J Biosci Bioeng. 2016 Feb;121(2):133-9
26140918
Bioengineered. 2016 Apr;7(3):189-97
27116572
Int Immunopharmacol. 2003 Dec;3(13-14):1715-29
14636823
J Biotechnol. 2008 Jan 20;133(2):201-7
17935815
J Biotechnol. 2011 Apr 20;153(1-2):22-6
21392548
Cytotechnology. 2012 Dec;64(6):613-22
22415736
J Biotechnol. 2011 Oct 10;155(4):377-86
21816183
Anal Biochem. 2011 Oct 1;417(1):159-61
21693100
Anal Bioanal Chem. 2015 Oct;407(26):8097-108
26329279
Biotechnol Genet Eng Rev. 2008;25:41-75
21412349
Mol Biotechnol. 2008 Jun;39(2):141-53
18327552