Hello all, I am planning to make a stable H1299 cell line with knockdown of my gene of interest. I understand that after 48-72 hrs of transfection, I should start the selection (with puromycin in my case). Here's my doubt, do I subculture and freeze the cells that I get after a week of selection (polyclonal) or do I split and dilute the cells to grow them in a 96 well plate to obtain monoclonal colonies?
I am planning to use these cells for overexpression experiments and mice experiment. Does the kind of follow up experiment affect the decision of polyclonal vs. monoclonal?
Another question I had was about adding antibiotic for selection. Is it a good idea to subculture the cells immediately after transfection and add the antibiotic at the same time? Because trysinization would weaken the membranes and might kill all the cells (?) Or, should leave the cells in the plate they were transfected in (no subculture) and start adding antibiotic in it?
Thanks!
Hello Pallevi,
Most standard protocols suggest waiting a few days before adding the antibiotic to allow the cells time to recover from the transfection, and also give them time to express enough of the antibiotic resistance gene. When we are making cell lines, we usually let the plates sit for a couple of weeks until all of the non-stable cells have died. What you then have is a plate that will have a lot of colonies on the plate, each colony is a monoclonal clone. We then pick the colonies and transfer to a 24 well plate. After culturing for a little while we then screen the clones to see what cell has the best expression, and that is what we use for protein production.
I have never tried using polyclonal cell lines, but I would think that you might have issues with variability overtime, especialy if the low expressing cell lines have a growth advantage over the high expressing ones. Additionally, often times you will have cells that are resistant to the antibiotic, but are not expressing your transgene. In a polyclonal cell line these cells will not be contributing to your protein yield and will just drop your average expression level on a per cell basis.
Good luck,
J
Hello Pallevi,
Most standard protocols suggest waiting a few days before adding the antibiotic to allow the cells time to recover from the transfection, and also give them time to express enough of the antibiotic resistance gene. When we are making cell lines, we usually let the plates sit for a couple of weeks until all of the non-stable cells have died. What you then have is a plate that will have a lot of colonies on the plate, each colony is a monoclonal clone. We then pick the colonies and transfer to a 24 well plate. After culturing for a little while we then screen the clones to see what cell has the best expression, and that is what we use for protein production.
I have never tried using polyclonal cell lines, but I would think that you might have issues with variability overtime, especialy if the low expressing cell lines have a growth advantage over the high expressing ones. Additionally, often times you will have cells that are resistant to the antibiotic, but are not expressing your transgene. In a polyclonal cell line these cells will not be contributing to your protein yield and will just drop your average expression level on a per cell basis.
Good luck,
J
Thanks, Dr. Finn!
It's more clear to me now. I think it would be wise to select monoclonal clones for my follow up experiments.
Pallevi
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