How to obtain a stable transfectant - (Aug/02/2005 )

Hi, all:

I'm trying to set up the transfection technique in my new lab.
Am using Clontech's pEGFP-C1 vector along with Lipofectamine2000 on 293T cells. Is it true what with the above reagents/materials, I could only get transiently transfectant, but not a stable line?

Initially, I would only want to see if the transfection itself works (by finding transfected 293Ts with green fluorescence). However, I'm planning on constructing my cDNA into this C1 vector and to obtain a stable line. Could anyone kindly advise as how I should approach to get what I wanted? Many thanks in advance!!

hi
when i got difficulties to obtain stable transformants, i made cell clones. I mean from a 15cm plate, i trypsin cells and resuspended them in 15ml medium. I put 25µl on a 10cm plate in G418medium (neomycin antibiotic resistance from my plasmid).
and 2 weeks after, cell colonies where enough big to pick them in 6 well plate. Basically, if you have transfected cells, you'll gona obtain at95% stable transfected cells.
fred

Thanks for your kind reply. If you don't mind my further following questions:

1. My vector also contains Neo resistant gene, in this case, when would you recommend that I add G418 to select my transfectant after the transfection is performed? And, I've read people seemed to use different concentrations of G418 (ranging from 0.5 to 1.5 mg/ml) for the selection purpose. Does it matter (in terms of G418 toxicity to the cells)?

2. Some people had said that to get a stable transfectant, you'll need to use retrovirus-based vector, since this vector would integrate into chromosomes, but not other vectors. The one I'm using is a GFP vector (pEGFP-C1 from Clontech, specifically). I can also obtain stable transfectant using this vector, I assume?

3. Once you got your stable transfectant, is it necessary to always maintain the transfectant under G418 at all?

4. Does it matter which cell lines to use in order to get stable transfectant? The cDNA I'm interested in putting it into a stable transfectant is a murine membrane protein.

Many thanks again!!

Dear Lab-Newbie,

Using the pEGFP-C1 vector with Lipofectamine2000 on 293T cells, you can easily produce stably transfected cells. You are correct in that the neomycin gene in pEGFP-C1 will allow you to stablilse using G418. With regards to the correct concentration, may I suggest you perform a G418 titration curve.

Seed cells into a multi-well plate (6, 12, 24, 48 or 96-well - what ever you have on hand). Use a density that will take 2-3 days to reach confluency (cover the entire well). Use media containing a range of G418 from 400 µg/ml to 1.5 mg/ml. Incubate your cells for a 2-week period, refeeding them twice a week. After 1 week, note which level of G418 has killed cells, and which level the cells are still alive. At the two week timepoint, note which wells have all dead cells. Choose the G418 dose that killed by week 1 and in which there were no surviving cells/ regrowth at 2 weeks.

Then, transfect your cells and at 24-48h later (when confluent), split them into plates or flasks (see note below) into media + G418. Remember to use a seeding density that will not be confluent again for 2-3 days (G418 takes 2-3 doublings to start killing, and cells need to be actively dividing). Refeed cells twice a week and grow until you have colonies and/or a confluent flask. These cells are now stably transfected cells.

Most people maintain the cells in G418-containing media. After you have plenty of freeze-downs/stocks, you can drop the concentration of G418 by half to maintain. If you are seeding cells for an experiment (not your stock flask), I often don't add the selective agent (saves money).

NOTE: if you would like to clone your cells, use a 6-cm or 10-cm plate for the selection. If you do not need to clone, use a flask (easier and less likely to get contamination).

I hope this helps and good luck with your transfection.

AussieUSA.

Dear AussieUSA:

Wow.. This is a very detailed and comprehensive instruction. Many thanks for your geneous help on lab newbie like me. Deeply appreciated!!