I have been trying to knockdown a gene of lysosome recently. When cells are transfected with pSuper-GFP-neo-target shRNA, the resulting transfectants seems hard to receive other plasmids for a second transfection via PEI. Worse still, the GFP positive cells seems to die after 48hr after the first wave of transfection. Can anyone give me some advice?
The knockdown of lysosomal proteins in some cancer cells have been known to be lethal. So I think your cells dying may be expected. The loss of a uptake-recycling mechanism may be expected to be detrimental.
Secondly, since uptake of plasmid involves endosomal uptake and importantly escape from that endosome to reach the cytoplasm, again it is feasible that this is blocked by interfering with lysosomal function.
Although PEI is know to promote plasmid escape from endosomes, maybe using a liposome-like reagent - lipofectamine or similar may work better than PEI since it forms a liposome to do the delivery. I am not an expert on the liposome mechanisms or anything, but it may work better than PEI.
1. Can you transfect cells with the plasmid ALONE? You may want t check if your plasmid is working in the first place by itself.
2. Can you reverse the sequence? Add plasmid first then shRNA?
Good luck !
I think the ancient method of calcium phosphate precipitation does not require endosomal uptake. This is described int eh original Maniatis Manual and still works pretty well in many cultured cells.
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