I want to stably transfect HEK293 cells with DNA and am wondering if it's better to transfect them while they're adherent or in suspension. Also, should I linearize the DNA?
Thanks!
I agree with Nicolas and Han-Jia. PEI is just as good as Lipofectamine 2000 for HEK293 cells, and yes, you should linearize your plasmid first if genomic integration is the goal. 293T cells, but not the parental HEK293 line, are neomycin-resistant, so if you plan on selecting with G418, you better make sure the cell line is identified correctly.
Lentis are very easy to work and yield stable transgene expression lines at higher efficiency than plasmid integration, so may be a better choice.
Lipofectamine works better with adherent HEK293 cells. Linearlzed DNA should have a better chance to be integrated into chromosome. However, if you want to make a stable transgenic line, viral vectors, such as lenti virus system, is highly recommended!
In our hands, there is not much difference in transfection efficiency between Lipo2000 and Polyethylenimine (PEI) transfection for the HEK 293T. So, I would recommend using PEI instead of Lipo200... you will save a lot of money! For your purposes, I would use the following setup:
Day 0: In a 6 well plate, seed 600000 cells per well
Day 1: Transfect 2ug of your mammalian expression plasmid using the method of your choice.
Day 3: Add selection agent such as Hygromycin B
Day 5: Change medium and add selection agent
Repeat until you can expand your cells. I generally switch my cell from a 6 well plate to a 10 cm and then to a 15 cm to make a stock of the stable cell line. In between, validate that your plasmid DNA was integrated by a conventional method (ie. induce your reporter or make a western blot), this way you'll have all the validation data on hands.
Good luck!
I agree with Nicolas and Han-Jia. PEI is just as good as Lipofectamine 2000 for HEK293 cells, and yes, you should linearize your plasmid first if genomic integration is the goal. 293T cells, but not the parental HEK293 line, are neomycin-resistant, so if you plan on selecting with G418, you better make sure the cell line is identified correctly.
Lentis are very easy to work and yield stable transgene expression lines at higher efficiency than plasmid integration, so may be a better choice.
You also might consider trying reverse transfection. This yields very high transfection efficiencies for 293s in my experience with Lipofectamine 2000.
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