[Study on influencing factors in efficiency of introducing gene into human keratinocyte (KC)].
OBJECTIVE
To observe the effect of plasmids in different size and gene transfection protocol on efficiency of introducing gene into human KC.


METHODS
Four plasmids in different size, inclu-ding pSUPER-enhanced green fluorescent protein (EGFP), pEGFP-N2, pHSER-green fluorescent protein (GFP) and ploxP-EGFP, were transfected into immortal human KC line (HaCaT) and human embryo kid-ney cell line (293FT) separately following transfection protocols of liposome (LTP), cation polymerizer (CPTP), electroporation combined with nucleus transfection agent (ETP) and lentivirus. 293FT was used as control. GFP expression was observed under inverted fluorescence microscope. The transfection efficiency (TE) was calculated.


RESULTS
(1) The four plasmids could be introduced into HaCaT (TE, 1.0%-3.3%) and 293FT (TE, 80.0%-84.7% ) following LTP. (2) The four plasmids could also be introduced into HaCaT (TE, 1.0%-3.7% ) and 293FT (TE, 81.3%-86.7% ) following CPTP. (3) Two shorter plasmids (pSUPER-EGFP and pEGFP-N2) could be introduced into HaCaT by ETP with higher TE than the othr two longer plasmids (pHSER-GFP and ploxP-EGFP), which were 22.3% and 19.0% vs. 4.0% and 3.3%, respectively. (4) pHSER-GFP packaged by lentivirus could be introduced into HaCaT with the TE reaching 97.0%, which surpassed the above three protocols.


CONCLUSIONS
It is difficult to introduce exogenous gene into human KC by LTP or CPTP; TE of lentivirus transfection protocol apparently surpasses
Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, the Third Military Medical University, Chongqing 400038, China
Li-Hua
LH
Wang
Dai-Zhi
DZ
Peng
Xin
X
Zhou
Jing
J
Liu
Yong
Y
Wang
Sheng-Dong
SD
He
Bin
B
He
Bi-Xiang
BX
Zheng
Zheng-Xue
ZX
Dong
D002460:Cell Line; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D015603:Keratinocytes; D008081:Liposomes / Q000378:metabolism; D010957:Plasmids; D014162:Transfection
D008081:Liposomes