I am working with third generation Lentiviral vector system and trying to transduce SiRNA for gene silencing. The problem I am facing is that the GFP sorted cells on FACS are showing 88-94% purity. Now when I culture them back and amplify the GFP +ve cells that are down regulated for the gene of interest, I find cells which are GFP-ve ( because of the sorting purity).
My concern is that when I would show downregulation by SiRNA, it may not be significant because of the untransduced cells. Any opinions about it?
Is this the purity normally can be achieved through FACS sorting and is it fine if we show downregulation even in the presence of GFP-ve cells.
Any help would be appreciated.
Thanks
Hi,
For the first part of your question, well it depends on the efficiency of your siRNA. If you manage to get a very good knock down of your interest gene, then ~10% of cells still expressing it at a wt level should not be a huge problem. But if you have a not-so-good level of knock down...
Anyway, the obvious solution is indeed seeking a better purity. I don't know every FACS machine, but on most of them (if not all modern machines) you can select a different sort mode. For example, on BD Aria facs, you can select the "yield" or the "purity" mode. The yield mode allows you to sort quite quickly, but at the expense of purity. On the other hand, with the purity mode you can achieve 99% of purity, but the sort will take longer.
The other solution to achieve a better purity is simply to lower the speed of the facs. The more events/s you have, the less purity you'll have in the end.
Hope it will be usefull for you!
Antoine
How many days after the transduction was it when you did the sort? A fraction of cells always gets only transient transgene expression due to the lack of integration, those will be losing GFP expression after a while. Ideally, you should culture transduced cells in a week or so before doing the sort, to let transient GFP expression fade. And for better purity use tighter gates, aim for the highest expression level.
Hi,
It is possible that the gene you are silencing is affecting cell growth and thus non-transduced cell take over the population.
Furthermore, does the GFP run off a different promoter then the siRNA? GFP promoter could be failing while siRNA is still being produced.
Does your vector have an antibiotic selection marker?
If you are getting over 90% green cells and you don't see you desired effect then you probably won't see an effect at 100% transduction.
The siRNA may not be working well. Before i go any further i would carry out a western blot for the protein of interest and compare it to control cells. Even if you have 60 - 70 % green cells you should see a significant downregulation of the protein.
If all goes well you other option if to seed single transduced cells into a 96 well plate and select a population of cells from 1 cell that was transduced with good efficiency.
One thing keeping in mind:
When you knocked down a gene/protein, after long-time culture, the protein expression might come back. So make sure to use early-passage KD cells or do western to check the protein expression in case that you cultured the KD cells for a relatively long time. Like a month. Good luck
Thank you all for the valuable suggestions. We have implemented some of the changes related to the FACS setup already. The sorting mode was purity with low flow rate. we gated stringently with the brightest population and mostly cells were healthy after sorting. So cultured them and hope it gets better now.
Only one more query about the comments of Rongqing Aaron Pan.
If using Lentiviral Cassette system, doesnt it down regulate the protein expression constantly. You mentioned it can come back. How is this? Is it about loosing transduced insert or the cell finds some alternate mechanism.
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