Inclusion of a reporter vector such as pCMV-GFP that allows assessment of transfection into your cells could address the issue whether the transfection agent and the quality of the DNA are optimal or not. Both reagents allow transfection to different degree in NIH3T3 fibroblast. However, nucleofection using an optimized program may greatly improve the outcome. Further, "selection" needs to be optimized.

I have already performed optimization for both reagents, and did a kill curve too...

Since none of the cells actually survived, it probably means that my vector of interest is not transformed into the NIH3T3 cells. Do you think there might be a problem with the transposase DNA?

Hi Jun,

On your concern regarding the NIH3T3 cells and the transposase, you can find proper answers and find appropriate solutions to your problem in a Patent, Publication number US20030143740 A1

Application number US 10/272,552, Publication date Jul 31, 2003 invented by Christine Wooddell, Hans Herweijer, Jon Wolff, entitled "Processes for transposase mediated integration into mammalian cells". The authors disclosed compositions and processes for transferring a nucleic acid into a mammalian cell (including the NIH3T3) utilizing a transposase to achieve nonviral integration of exogenous nucleic acid into the chromosomal DNA of the cell.

Best wishes,

Ilya

HI Jun,

it's been two years since you posted this. Were you able to find out what your issue was? I am about to try to perform a transfection into 3T3 cells myself, so I'd love to hear how you solved the issue.

Best,

Anna