I'm getting rather desperate now with continually failing to obtain stable cell lines. My cells just all die when i add selection agent, even though i tested with a kill curve at a range of cell dilutions going to very sparse cell densities prior to selecting the concentration. I have tried to be gentle with the cells, waiting until they divide several times before adding selection, adding some conditioned media, extra FCS, since they do not thrive when isolated. I am using BJ-5ta cells (TERT immortalised human fibroblasts). They are maintained on hygromycin to maintain their immortality, via TERT expression. I am therefore using puromycin for selection of my genes of interest. I am transfecting my gene of interest in a Tet-One inducible expression vector together with a linear puromycin selection marker at a ratio of 20:1, as recommended. I am also transfecting DsRed in parallel, so i can actually monitor transfection efficiency and see if cells are dividing and passing on expression of the gene. I use X-fect for transfection, which gives very little mortality, unlike lipofectamine. I know i have lower transfection efficiency with my Tet-One construct than DsRed alone, such that there are too few cells for me to do experiments with transient transfections. i get very high transient efficiency with DsRed alone, but still no stable cell lines. I wonder several things... is genomic integration so low that i just need to transfect more cells to be lucky enough to get one incorperating the selection marker (at 1:20 concentration of GOI), and i just need to scale up? I have the feeling some of my transfected cells might not be as healthy as the untransfected ones, and are less likely to divide. I am open to any kinds of tricks and tips from experienced stable cell line generating researchers, that i may not be aware of (conditioned media? how soon before addition of antibiotic?, how often should i change media?) I also tried seeding transfected cells to 96 well plates to try to get single clones, however i have a lot of trouble with this. I dont manage to get approx 1 cell per well.. even if i count my cells, i think they sediment prior to pipetting, or they get damaged by too much pipetting, and then they dont like being alone in the wells (so i add some conditioned media), but I really have trouble to see if i have one or any cells in the wells of a 96 well plate anyway.
The Tet-One plasmid is from Clontech. It is a single plasmid with the tet activator and promotor for gene of interest all on the one gene. I'm not sure i understand your question.
i do not know expression system from Clontech (Invitrogene now) where tetRE and the target gene is in the same plasmid. technically it is possible. but they sell clones of different cell lines with tetre and a plasmid with promoter. i just check it on the website. maybe i am wrong.
Dear Christa, genomic integration being a random event, you need to transfect as much cell as possible to get "lucky" as you said.
My suggestion try another transfection approach such as Magnetofection (http://www.ozbiosciences.com/content/13-magnetofection) which should lead to higher DNA delivery and more chance to get genomic integration. This technology will also allow you to perform two sequential transfection such as day 1 and day 3 which should improve the generation of stable cells
Hope this can help
Hi Christa, I agree with Olivier, your low transfection efficiency might be the problem. I use Lipofectamine 3000 from Life Technologies. It has very low toxicity and high transfection efficiency. Once you get higher number of cells integrated with your insert, you can start adding the selection medium and change it every 3 days.
Hi used Lipofectamin 3000 and nucleofection with site specific insertion systems and I always get minimum of 10 new clones.
Few suggestions: as already mentioned, choose a better transfection/delivery system.
try site-specific integration systems over random integration. In random integration systems, many variables are added to your experiment at the molecular level. A) integration into the silent sites of the genome, B) integration and excision of the integrated gene due to over expression of enzyme, and etc.
If you have no other choice rather general/random integration, maximize the amount of DNA according to 6-well plate protocols. and then expand your cells with 1:10 ratio three days post transfection. at day 4 you can start selection by adding the selection marker. wait for two to three weeks to see the new cells.
hope it help
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