I am trying to establish a stable cell line expressing a gene of my choice using a prion protein knock out cell line, Lipofectamine and Zeocin as selection antibiotics.
Not ruling out the fact that my handling of the cells might have not been near perfection, the transfected cells consistently responded to selective treatment 8-10 days but after ward began to grow at sporadic rate whereas they are expected to reduce at steady rate due to the bactericidal action of the antibiotics and after some period of period begin normal growth. What could be responsible for their observed sporadic growth rate?
number of cells and their adhesion to each other can influence growth rate. Also secretions into the media can affect growth. As Ali suggests I'd refresh the media regularly (adding fresh antibiotic and getting rid of secretions) and/or transfer the cells (this adresses cell number and adhesion in addition).
As mentioned, it is completely normal that after the selection is complete the cells that have a stable knock-in (or rare resistant cells) will grow at normal rates. All they have to do is produce the antibiotic resistance protein and then they are able to resume normal growth. If your whole cell population is growing at this normal rate and you have your gene stably integrated (as observed by PCR or Immunoblotting) than you should have no concerns...just keep the Zeocin in the media. However, if you didn't notice a dramatic dying off of cells during that 8 days, and haven't replaced the media with fresh antibiotic, it is likely that you aren't 'selecting' the cells effectively enough. Did you do a 'kill curve?' Did all your controls die while the transfected cells are still dividing?
Thanks Prof Erik Martin
Yes, all cells in the negative control ( with only transfection reagents and no Target DNA nor Vector DNA) plates all died after 8days. Am only concerned about the growth rate. The cells confluences on a 100mm plate in less than 48hours of passaging even when splitting is done at 1:10 ratio
Joshua, you are welcome. After reading your latest comment, it does sound as if your cells are dividing at an abnormal rate. Do you think the gene you are stably expressing in them is speeding up the division rate? What cell type are you using? I find this interesting and would like to learn more in case I encounter such an event in the future. Thank you.
@ Dr Ali, l did the selection and growing of the cell line on liquid medium- DMEM/ 10 % FBS/ 1% Penicillin, Streptomycin
@ Prof Erik, am using a prion protein knock out cell line- Zpl 3-4, trying to stably transfect Chinese Water Deer Prion Protein into it. Although when l reviewed some journals on effect of genetics- prion protein polymorphism on Chronic Wasting Disease l discovered Polymorphism could affect cervids' susceptibility to CWD, PrPSc characteristic, doubling time and replication in vivo but not sure if it will be the same scenario in vitro.
Joshua, I'm unsure if you have considered a lentiviral gene delivery system or not, but the transfection method of gene delivery can be a bit unstable and is not always reliable for long term studies of your transgene. Your cells may have variable growth rates due to this unstable gene that was transfected into your Zpl 3-4 cells. A word of warning however, retro-viral vectors need proliferative cell lines for optimum expression; this thereby limits this delivery method in certain cells lines invivo.
Also, how was the growth rate before transfection? Some cell lines do grow quite well in 10% FBS, so if you cut back the FBS to 5% you should see the cells growing a bit slower; this way the cells will be much easier to manage. If you haven't already done so, preform a kill curve on your transfected cells. Would you mind telling me what antibiotic are you using to select?
One more thing, if the growth rate is different than before the transfection process, have you considered the possibility that your Spl 3-4 cells are contaminated with cells of a different lineage? If your cells became contaminated just before your transfection process, that would explain the initial variability in growth rates too.
Hope all this helps! Let me know if you have any questions.
@ Gabriel R Anderson. The cells reduced first 8 days of antibiotics selection and started growing at an increasing rate after this time. I am using Zeocin for my selection.
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