[Optimizing condition for oligofectamine-mediated SP1 decoy oligodeoxynucleotides transfection into SV-40-PED cells].
OBJECTIVE
To determine the optimizing parameters in transfecting the SV-40-PED cells mediated by oligofectamine.


METHODS
With a change of Decoy oligodeoxynucleotides (ODNs)/oligofectamine in ratio and the transfection time, the uptake rate and the mean fluorescence intensity of SP1 ODNs in the SV-40-PED cells were measured by flow cytometry to evaluate the transfection efficiencies. 4 microl oligofectamine with different concentrations of ODNs(2.5, 5.0, 7.5, 10.0 and 12.5 microl) were put into 100 microl of DMEM without serum and antibiotics. the (SV-40-PED) cells were transfected after 20 min at room temperature. the final concentration of SP1 decay ODNs were 50,100, 150, 200 and 250 nmol/L. Transfection effieiency was detected at 26 h after transfection. The intracellular distribution of SP1 ODNs was determined with a fluorescence microscope. The lactate dehydrogenase (LDH) activity in the supernatant was measured to assess the cytotoxicity.


RESULTS
The uptake of SP1 ODNs into the SV-40-PED cells was significantly improved by oligofectamine. The cell appearance did not change much in the groups of 50, 100 and 150 nmol/L. In the groups of 200 and 250 nmol/L, the cell reverted after being shrinked and altered to round. At 26 h after the transfection, there was no marked change in the cell form at the concentration of 250 nmol/L. There was floatation at 48 and 72 h after the transfection. Under the fluorescence microscope, we observed fluorescent materials distributed in the cell nucleus in the successfully-transferred groups. We could see the nucleoli clearly in the groups of 200 nmol/L and 250 nmol/L. There was a stronger fluorescence intensity with a higher concentration and the fluorescent materials gathered at the cell nucleus. At the final concentration of 250 nmol/L, the LDH level was 137.12+/-3.92 U/L in the 72-h group, which was significantly higher those that in the 26-h group (49.61+/-17.13 U/L) and the 48-h group (120.26 +/- 8.42 U/L) (P<0.01). At 26 h after the transfection, there were no statistical differences at the above LDH levels in the different-concentration groups (P > 0.05).


CONCLUSIONS
Transfection efficiency is the highest when the final concentration of the SP1 decoy ODNs is 250 nmol/L during the incubation of for 24 h in transfecting the SV-40-PED cells.
Huang
Yabing
Y
Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhang University of Science and Technology, Wuhan Hubei, China. hyb3022@163.com
Xu
Jing
J
Chen
Song
S
D000818:Animals; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods*; D006084:Graft Rejection / Q000517:prevention & control; D008055:Lipids / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000008:administration & dosage*; D013552:Swine; D014162:Transfection / Q000379:methods*
D008055:Lipids; D009838:Oligodeoxyribonucleotides; C484027:oligofectamine