I have the following problem. I am looking into a gene, which I've stably overexpressed in an AML cell line. This overexpression leads to increased colony formation capacity as measured with CFU assay. But when I try to support this with a BrdU assay, the overexpressing cells show less proliferation (% of BrdU pos. cells) and a bigger G1-population, compared to the empty vector control. Both cell population showed 95% viability prior to the assay. Can you think of a reasonable explanation for this?
If I've got the question right, the increase colony forming mutants get arrested at G1-S transition, whereas WT cells can divide, and both of them had 95% viability.
Keeping lab-errors aside, the reasons for arrest at G1-S transition of the specific cell line means that these cells cannot complete (have an obstacle at) this transition. Right now I'm looking for a possible answer, but please provide a feedback whether I've got your question right.
How long do you incubate with BrDU? Long incubation (over 24h) are not recommended!
Thank you very much for the replies! I incubate for an hour, but I am actually thinking of reducing that..
Dear Elbay: yes, that is the questions. The transduced cells (that form more colonies than the control population in the CFU assay), appear to have a G1 arrest and are not as proliferative as their control in the BrdU assay.
A possibility:
the gene,responsible for extensive colony formation inserts BrdU; this may compromise mRNA production/translation. So less protein (or peptide) responsible for extensive colony formation will be produced. Checking the mRNA level after incubation with and without BrdU will be helpful.
Another method is looking on Ki-67 (a protein produced in proliferating cells) expression.
If that is the case, if the inserted gene contains many thymine residues, then, it is possible that, at the G1-S transition state, the bromine residues (that was incorporated in place of thymine residues) could be catched by the repair system, and the result of repair system (when incorporating normal nucleotides, sometimes not all true nucleotides are inserted) may block the gene function. and lead to G1-S blockage. That is of course one of the many possible results. Also, as mentioned above, try to control other markers of cell division, like Ki-67.
Hope that helps.
Good luck!
Thank you all very much!I think I ll try the Ki-67 assay and keep you posted on the results!
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