THP-1 transfection with Neon for CRISPR

[Mohsen Honarpisheh]

Dear all,

Does anyone succesfully generate THP1-KO cells?  We are currently doing transfection on THP1 with PX458 plasmid. Since THP1 is very diffcualt to do transfection due to cell death. We optimized the condition for transfection by decreasing voltage and increasing pluses and time. We are able to get alive cells by more than 80% but with less transfection efficacy.  

Does anyone know how much efficacy would be enought to do sorting to getting single cell?

Best,   

[Martin Zhu]

Hi Mohsen,

We have not CRISPR'ed THP-1 cells, but we've tested transfection methods for THP1 cells extensively, including various lipid-based and electroporation-based ways.

Electroporation is really not the way to go. We tested Lonza's recommended Nucleofection kit and our homemade buffers under different electroporation protocols. The best result is about 40-50% efficiency (with pMaxGFP from the kit). In each test, over half the cells would die.

After careful juggling between DNA and lipids doses, the one that finally worked was TransIT-2020 from Mirus Bio. Use 10ug DNA and 30uL TransIT-2020 per 5 million cells. Incubate the cells with DNA/TransIT-2020 in OPTI-MEM for 5 hours and replace with complete medium. The strong expression (a FLAG fusion protein in our case) of DNA vector is detectable after 48 hours.  Cells are almost 100% alive with this method. You can incubate the cells in DNA overnight, but we found a lot of cells would attach to the culture surface, although the viability do not seem to be affected at all.

FYI, the following didn't work well in our hands: Lipofectin, Lipofecatamine 2000, Lipofecatamine LTX, TransIT-Jurkat, TransIT-X2, TransIT-LT1, and FuGENE HD.

Anyway, I believe you should be able to get PX458 delivered by TransIT-2020.

Best luck to your experiment.

Martin

[Mads Munk]

Hi Mohsen

Which settings did you use with the Neon machine?

BR Mads