Usually, I use lentivirus transduction of shRNA for knocking down of my target and after producing of stable cell line and take my first replicate the knocking down efficiency begin to decrease with RT-qPCR from the second replicates.
Can anybody help me to solve this problem?
Thanks
It is good to keep low concentration of selection antibiotics in the culture all the time. While, the compensatory response inside the cells and the natural selection of population with high proliferation rate may gradually decrease the knockdown efficiency. Freezing several vials as early as possible and using the early passages will be a good option. Otherwise, try inducible method as suggested by Dirk.
Thank you very much Fei Wu
usually i use 2 at the same time and the kd efficiency in the first subculture over 80% then in the 2nd subculture decrees to 60% and the 3rd become 30% and the 4th no difference
Limiting dilution and single cell cloning after a period of 2 weeks can help picking integrated virus clone and stable knock down.
I agree with Goodwin: single cell cloning can help you find the best clones.
If your gene knockdown is deleterious for the cells, you will always be hindered by cells automatically being selected for a low knockdown. In that case, you can choose an inducible shRNA expression system, or use clones for only a limited time, then throw out the culture and go back to an earlier vial.
It is good to keep low concentration of selection antibiotics in the culture all the time. While, the compensatory response inside the cells and the natural selection of population with high proliferation rate may gradually decrease the knockdown efficiency. Freezing several vials as early as possible and using the early passages will be a good option. Otherwise, try inducible method as suggested by Dirk.
HI there,
If you need a stable KD line, the limiting/sigle cell solution is probably the way to go, however working with a clonal line does always rise some doubt on the general significancy of your results. In addition it could take months until you get enough cells for your experiments... In addition if your KD is so deleterious for the cell growth you risk to select clones that have a compensatory mutations... finally it seems to me a waste to use a viral delivery system and then pick single colonies.
The easy explanation for your observation is that you have an incomplete selection after viral transduction so the few not infected cells with the time overgrow the majority of the trasduced one. You do not mention the antibiotic, the concentration and for how long you treat your cells, but I would start working with this parameters and as suggested by Yunjian, I will keep some antibiotic in the culture. Of course it could also be that you get two separate integration for the hairpin and the resistance then you will always select the ones that are resistant but do not contain the shRNA...
Looking at the positive side, it seems to me that you have a clear phenotype for your knockdown, it seems to affect the cell growth!
Hope this help.
Good Luck
Max
I agree with the proposals that have been discussed , it is especially important to choose a lower concentration of antibiotic maintenance , selection of individual clones and freeze vials. Another option , besides using CRISPR cas , is to use constructs with GFP and once achieved the clones do sorting and grow them .
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