I have found that it is harder to induce a large volume of procyclic cell culture (>50ml), compared to a normal 10 ml culture. By 'harder to induce' I meant that percentage of cells showing the expected RNAi phenotype is significantly lower. The concentration of cells are at similar levels, and Tet is always made fresh. I wonder if this has any basis? Or is this a common phenomenon? Thanks!
Hi Charlie,
I've not experienced change in phenotype across different culture volumes (cell density might influence certain phenotypes, but I'm not sure how volume could). However, I have seen loss of RNAi phenotypes over time in cultured bloodstream form T. brucei, even in the absence of prolonged induction. This is probably due to low level 'leakiness' of the inducible system, leading to selective pressure against retention or expression of the RNAi cassette. I saw this with a series of histone and histone chaperone RNAi cell lines, induction of which should give a dramatic phenotype over a couple of days.
Did you do your initial induction in 10 ml, keep some uninduced cells growing for a time, and then repeat the induction in a larger volume using the uninduced cells from the previous experiment? If so, this might have provided enough time for suffiencient selection to elicit sequence or chromatin changes in your inducible RNAi cassette. I solved this problem by going back to a fresh set of clones that I'd frozen down soon after identifying the original transformants. Hope this helps.
Sam.
Hi Sam, thank you for your generous help. I have considered the possibility of 'leaking' before and it indeed could be the case. Yes I induced 10ml first and then scaled up, but I have tried going back to a 10 ml culture after the unsuccessful trial with large volumes and it seemed to be working fine again. It might be that I did not manage to control the density well enough for the big volume. Anyway thawing a new tube of cells definately worth trying! Thanks again~~
Hi Charlie,when you increase the volume, make sure that the ratio surface/volume is unchanged, otherwise access to oxygen will be modified and this will impact on cell behaviour. I am not sure whether it would modify the tet-induction but that's a common factor for variation. Good luck with your experiments!
I've seen the loss of effect upon induction in 2913 RNAi procyclic cultures that were kept in culture for more than two weeks. As Sam has suggested, taking a new stabilate solved the problem. Since then, everytime I want to do RNAi assays I use a fresh aliquote every time, inducing at the same passage for reproducibility. Never saw differences in scalling up, but as Phillip suggested, the ratio surface/volume is a factor to take in account, specially because the procyclics grow in closed flasks, so I just use bigger flasks considering the volume needed. Hope you've overcome this problem!
Hi Catarina, thanks for sharing your experience~! I also started to revive new alliquotes every time I needed to induce. Just a follow-up question: Usually how many passages after thawing will stablize a 2913 RNAi cell line?
Hi Charlie, I use usually by passage 4. So, take them out (P0), passage 1:10 twice (P1 and P2), then passage them to 5.5x10^5 cells/ml (P2), follow them up to see if they are growing ok, when at 1x10^7cells/ml passage again to 5.5x10^5 cells/ml (P3). When at 1x10^7cells/ml I just set up the parent cultures for the RNAi assays (P4). I find that at early passages (P1 and P2) they don't behave normally (eg. the cell cycle profile by FACS doesn't look normal), so I've always used them by P3/P4. Has the new aliquotes worked for you then? Hope it has!
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