I knockdown a gene by shRNA (lentiviral) in human iPSC. The shRNA construct is GFP tagged. What I observed under the microscope was, the GFP+ cells (the ones with shRNA, hence that gene of interest getting knocked-down) were dividing more/faster compared to the control. I did cell cycle analysis on that KD with the control and it turned out that the KD sample has almost double the percentage of G1 cells! Moreover less cells were in S and G2/M phase cells in comparison to the control. In the first place it looks like the cells are having a G1 arrest, which does not fit to what I expected, because the cells were growing faster. And also ESC are known to have short G1, and I expect iPSC to behave similarly. Since G1 arrest does not explain it, I was wondering whether the cells might have quicker S and G2/M phase upon knockdown of that particular gene, which might make it look like they are accumulating in G1 as if under arrest? I know one could induce arrest at different phases in various ways, but would it be possible to change the length of the phases, and eventually the relative distribution of cells throughout the cell cycle?
If KD cells are more proliferative than control cells, the time it takes to change from G1 to S becomes longer after the gene of the interest was silenced. Why not check the cyclin E expression, which increases when they undergo progression to S phase, whereas decreases when they stop at G1/S checkpoint via AMPK/p53-p21 axis???
Dear Ceren,
Check if the gene you are knocking out, if it is a tumor suppressor gene, it explains the faster cell division.
Best,
Mannan
Hi,
in principle you can shorten S and G2 phases (see attached papers). To find out what happens in your cells, what about a good, old-fashioned analysis of doubling time of your knockdown cells, to check whether they actually divide faster.
If you want to measure the length of cell cycle phases, you can do cumulative EdU labeling. With cells in vitro and a flow cytometer, this should be not a big deal.
Hope that helps
Article Neural stem and progenitor cells shorten S-phase on commitme...
Article The CDC25B phosphatase shortens the G2 phase of neural proge...
Article S-phase duration is the main target of cell cycle regulation...
Thanks for the papers and comment, Christian! I will check the cell cycle length as you suggested.
@ Mannan, tumor suppressor could have explained it but it is not a tumor suppressor gene.
@ Go, I am not clear about why the cells should have a longer transition from G1 to S when they divide faster?
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