[Stably transfection of DNA to human fibroblasts: a comparative study of three approaches].
OBJECTIVE
To search for the best approaches to transfect plasmid pEGFP-N1 into human fibroblasts.


METHODS
Cationic polymer mediated transfection, cationic liposome transfection and electrotransfection were compared for transfecting plasmid pEGFP-N1, a plasmid that expresses G418 resistance protein and Enhanced green fuluorescent protein (EGFP), into Human fibroblasts. The expression of EGFP was observed 24 hours after the transfection by fluorescence microscope. The transient transfection efficiency was measured by flow cytometry. The stable transfected cells were screened by G418.


RESULTS
Electrotransfection had the highest transient transfection efficiency (56.8%), with the most masc clones of stably transfection and the least time taken to form big masc clones (20 days). Cationic polymer mediated transient transfection had the lowest efficiency(1.7%), with the least masc clones and the longest time taken to form the big masc clones (30 days). Cationic liposome transfection had a transient transfection efficiency of 39.9% and took 25 days to form the big masc clones.


CONCLUSIONS
Electrotransfection is the best way of stably transfecting plasmid pEGFP-N1 into human fibroblasts.
Lu
Xiao-yan
XY
Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
Xie
Hui-qi
HQ
Li
Li
L
Zhi
Wei
W
Chen
Xiao-he
XH
Deng
Li
L
D002478:Cells, Cultured; D018274:Electroporation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D052816:Foreskin / Q000166:cytology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D008081:Liposomes / Q000737:chemistry; D008297:Male; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D008081:Liposomes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins