Subject: Stable Transfection Trouble Shooting
I am electroporating several plasmids into an already CRISPR modified Cancer Cell line. All the cells look fine post electroporation. However, once I add selection marker boom they are dying, I have gone really low with the antibiotic that I actually titrated.
Cell Viability post electroporation - 60% analyzed by Flow Plasmid integration into the cell - 50%, analyzed by flow for RFP
My thoughts - there may be transient transfection in these cells, however, no stable transfection
What I am trying - different plasmid concentrations up to 20 ugs, for Electroporations - I am using Nucleofector, even lipid-based transfections (here the transfection efficiency is super low around 5% - but cells die after antibiotic treatment). We are in process of preparing lentivirus, but we would like to avoid this method as much as possible.
IMP - all these plasmids have two genes separated by IRES and one selection marker, antibiotic is acting in the same way in both modified and parental cells, so I am guessing the CRISPR is not a problem by itself
Feel free to pour in your thoughts, anything would be great Have a great day
"once I add selection marker boom they are dying"
This is normal. Even if you get a very high transfection/electroporation efficiency, only a very small number of cells will actually integrate the plasmid into the genome (something like 1/10^4 - 1/10^5 is typical, depending on cell line and plasmid).
So as the non-integrated cells divide, the electroporated (non-integrated) plasmid gets diluted out, resistance decreases and the cells die. Once the mass die-off is complete, you should start to see single-cell-derived colonies start to form. These are cells which have the plasmid integrated, and therefore they replicate the resistance gene as they replicate their own genome. Typically I find that you will start to see colonies at around 7-14 days, depending on the antibiotic and growth rate of the cells, and how happy they are as single cells.
If you get what look like resistant single cells that won't grow quickly (or at all), you can try growing them in a 50/50 mix of fresh media and 24-hour conditioned media from a full flask of your cell line, supplemented of course with whatever antibiotic you are using. This will help to provide some of the autocrine growth factors that your cells use. If you have already CRISPR edited these cells, I guess you shouldn't have an issue with this, unless your edit affects proliferation/single cell growth.
"once I add selection marker boom they are dying"
This is normal. Even if you get a very high transfection/electroporation efficiency, only a very small number of cells will actually integrate the plasmid into the genome (something like 1/10^4 - 1/10^5 is typical, depending on cell line and plasmid).
So as the non-integrated cells divide, the electroporated (non-integrated) plasmid gets diluted out, resistance decreases and the cells die. Once the mass die-off is complete, you should start to see single-cell-derived colonies start to form. These are cells which have the plasmid integrated, and therefore they replicate the resistance gene as they replicate their own genome. Typically I find that you will start to see colonies at around 7-14 days, depending on the antibiotic and growth rate of the cells, and how happy they are as single cells.
If you get what look like resistant single cells that won't grow quickly (or at all), you can try growing them in a 50/50 mix of fresh media and 24-hour conditioned media from a full flask of your cell line, supplemented of course with whatever antibiotic you are using. This will help to provide some of the autocrine growth factors that your cells use. If you have already CRISPR edited these cells, I guess you shouldn't have an issue with this, unless your edit affects proliferation/single cell growth.
My thoughts: 1) Use lentivirus approach (even though you don't want to). 2) Use an inducible expression approach (preferably for lentivirus, but if you cannot do, using plasmids). #2 will take care of selection pressures against expression of your GOI.
It maybe as Philip states, you have some stable cells but they will take time to grow out, 7+ days, which is usual in our lab.
Since electroporation and the transfection/expression of foreign DNA stress the cells out, you may well find that they require lower antibiotic levels initially found in titration experiments, due to the cells being under greater strain and therefore more sensitive. This is especially true if the gene expression is not controlled.
My thoughts are:
1. Leave the cells post transfection a little longer to recover, 24+ hrs, then add antibiotics.
2. Do not be too hasty to throw cells out. Analyse cells +7 days just in case
3. Run controls: Plasmid with same resistance marker, perhaps empty plasmid or containing another irrelevant gene.
4. Carry out titration of antibiotic resistance on cells that have been electroporated (+/- irrelevant plasmid, different resistance marker). Do you get the same results as previous?
5. Do non transfected cells recover post electroporation: is the method you use sound.
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