lentiCRISPRcas9v2 Plasmid transfection kills cells.

I am trying to use lentiCRISPRcas9v2 and have designed 6 different guides (6 different plasmids) in a canine osteosarcoma cell line.

We have designed 6 guides to target multiple exons on gene of interest we want to knock out as just one plasmid targeting the in the first exon didn't work (still expressed protein)

We are using lippofectamine 3000, with plasmid and guides waiting 48 hours removing media and adding puromycin for selection.

We have conducted puromycin toxicity assay for the cells before hand to find the right dose.

However every time we try to do this, either with just one, two or four plasmid constructs it kills 100% of our cells.

Even the control with just the original entiCRISPRcas9v2 plasmid is 100% dead

As we plan to do in vivo experiments we want the cas9 expression to be short lived and not permanently expressed and therefore are not competent cells or viruses.

I really hope someone can provide some advice.


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Fuchsia Kelpiealmost 5 years ago

Do you know if lipo 3000 can effectively transfect plasmids into your cell line? Have you transfected a GFP control to test this out? If not, you can try nucleofection, lipo 2000, PEI, or other transfection reagents.

I have heard of 1 instance when someone has had the Puromycin in the vector recombine out during plasmid prep, so you might want to sequence that region to make sure the puromycin resistance gene is still intact in your plasmid.

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