tranfection of 3T3-L1 cells post differentiation - (Jul/30/2007 )
I am new to the forum. I am having problems in transfecting the 3T3-L1 cells after differentiation. I used electroporation till now. I want to transfect pre-miRNAs. Anyone have any experience regarding this???
Please do suggest me some better methods or improvement of the electroporation....
I have worked with 3T3-L1 cells before and they do not transfect easily, especially after differentiation. We were attempting transfections with fugene, lipofectamine, polyfectine...ect. I think we tried just about every commercially available reagent but were unable to get above 10% efficiency. We turned to using adenovirus and a stable 3T3-L1 cell line expressing the coxsackie virus receptor (CAR). This was the only way we could obtain a reasonable expression level in these cells. Otherwise you may want to think about creating a tetracycline controlled stable cell line that can express the pre-miRNAs you desire. I know both of these methods are a lot of work and will take more time than you want but I was never able to successfully transiently transfect these cells. Sorry.
I'm currently working with these cells, and as said rkay447, they are a really tough line to transfect. I also tried to transfect with fugene, lipofectamine and electroporation, but none worked. Adenovirus transfection of 3T3-L1 not expression the CAR is really hard to achieve, i.e. MOI of 2000 to get about 40-50% of efficieny.
We've been using retroviruses and lentiviruses to infect cells with shRNAs and it worked like a charm. We often get 100% efficiency with this method.