I made a stable cell line of a GFP fused gene which are resistance to hygromycin B. Cells in the first few passages were nearly 80-90% expressing GFP as analysed in the microscope. But after nearly 15-20 passage, I checked again in the microscope and I can hardly find GFP expressing cells, while still resistant to my selection medium including Hygromycin B. Is it possible that the cells can only keep the hygromycin B resistant feature without expressing my target gene?
You may better isolate several single clones. When you take the whole pool, you will have cells stably expressing GFP but also cells with integrates that do not express GFP This may be due to an integration in an inactive locus or due to an integration where GFP is not properly inserted. In addition you may have individual integrations that undergo silencing and in case of tandem integration you may loose some of the integrates due to recombination. There may be also recombinants with reduced growth.
Certain promoters tend to get silenced over time especially if the gene being expressed was toxic to cells. We experienced similar problem in Jurkat T cells when the cells were transfected with a vector expressing a scFv under the control of CMV promoter. We eventually cloned our gene in a different vector that expressed the gene under EF1 Alpha promoter and managed to get a stable cell line. Certain clones can still be unstable and therefore passaging them for several generations may not be a good idea.
You may better isolate several single clones. When you take the whole pool, you will have cells stably expressing GFP but also cells with integrates that do not express GFP This may be due to an integration in an inactive locus or due to an integration where GFP is not properly inserted. In addition you may have individual integrations that undergo silencing and in case of tandem integration you may loose some of the integrates due to recombination. There may be also recombinants with reduced growth.
Hello
i agree with researchers .
look at links here , i hope help you
Good Luck
http://www.flyrnai.org/DRSC-PRL.html
http://www.sciencegateway.org/protocols/cellbio/cell/stable.htm
http://groups.molbiosci.northwestern.edu/morimoto/research/Protocols/II.%20Eukaryotes/C.%20Stable%20Cell%20Lines/2.%20Gen's%20protocol.pdf
https://www.mirusbio.com/tech-resources/tips/generate-stable-cell-lines
http://www.origene.com/assets/Documents/HuSH_29_stable_transfection_GeneDex.pdf
http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_BenchGuides_Guideline_for_Generation_of_Stable_Cell_Lines__Technical_Reference_Guide.pdf
I would also add that 15-20 passages is a long time to keep cells out in culture, a lot can change during that time and increase variability in your experiments. A good idea for stable cells is to make sure you use IRES based plasmids as this means that cells will only survive if they express the resistance gene and your tagged protein of interest.
pIRES-hygro9 and pIRES-puro are good in my hands.
good luck!
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