We have been unable to transfect stem cells using a protocol that has been in existence in our lab since 2014. Essentially all the cells that take up plasmid appear to die. This is what we are trying to do to ascertain the cause
1. First we checked that the plasmids we are using (dsRED; YFP; CRISPR guides) are intact and showed very high expression of fluorescence in HEK cells with lipofectamine. In addition, all our CRISPR pX330 and px459 guides show successful cutting in these cells indicating that they are intact.
2. Upon transferring these to our iPSC line we transfected as per the conditions in the attached protocol:
0.8x 10 (6) cells per electroporation condition, diluted in 12 ul of Buffer R then added to 1ul (~600ng) of plasmid, or 1ul of elution buffer from the midiprep kit we use (we use a low endotoxin midiprep kit from Invitrogen), but have also acquired plasmids from our colleagues overseas) . This was electroporated in a 10ul neon tip using the following conditions: 1400; 20;1, and subsequently seeded into E8 medium containing ROCK inhibitor in 6 well plates.
3. 24 hours later
Cells exposed to the elution buffer only (no plasmid but still electroporated) seeded beautifully with minimal cell death.
Cells seeded with plasmid (all of them) experienced severe cell death but some YFP was observed in live cells. Mostly the fluorescence in YFP and all the dsRED that we observed was in dead cells.
4. 48 hours later:
Cells exposed to the elution buffer only (no plasmid but still electroporated) were still superb.
Cells seeded with plasmid (any of them) show >95% cell death. The only fluorescence we see is in dead/ floating cells.
It's something we robustly have worked on in our lab fir 5 yrs.
I have done the following:
a) different plasmids (from different countries)
b) different kits (including 10ul and 100ul; new and old)
c) imaged the stem cells for mycoplasma - all clear
d) bought a new pipette gun (ours was getting stuck)
I've tried to electroporate HEKs but also had poor survival rates. Essentially the only thing that is consistent with our experiments is that any cells that appear to take up plasmid die.
Thank you, Charles D Anderson
for your response.I also thought it might be a plasmid toxicity problem but we received a RFP plasmid with no endotoxin and transfected the iPSCs but the same result was observed as with the other plasmids.
Shivani
Chne Jing
, thank you for the response.I have been growing cells between 80-90% confluency before transfection.
Cells transfected with any form of plasmid (endotoxin-free, low endotoxin, maxiprep) die, whereas cells electroporation with the same buffer the plasmids are resuspended in survive.
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