Lentivirus transfection using RAW 264.7 cells - (Jun/02/2014 )
I did a RAW264.7 transfection using lentivirus with a pLKO.1-based plasmid for RNA interference. I have listed my protocol below. It didn’t work. Which part of the protocol should I change in order to make progress?
In addition, if the lentiviral transfection efficiency is truly low, which is the case for RAW264.7, are there any alternative approach which I can do to knock-out or knock-down a particular gene in it.
1 plate RAW.264.7 in a 12-well plate at a density of 1E6 cells/well, incubating for 3 h
2 mix the appropriate volume of the viral stock solution and polybrene
- didn’t measure the viral titre beforehand
3 add 1 mL of viral mixture to the wells. After 4 h, add an additional 1 mL of cell growth media. Incubate the cells for an overnight
4 next day, transfer the cells into a 6 well plate and directly going into the puromycin selection process <6 ug/mL>
5 After 1 day, transfer the cells into a 10 cm^2 dish. Perform an additional 1 day of puromycin selection process <6 ug/mL>
1 nearly 99 % of cells dead. The remaining 1 % cells appear to have higher resistance toward puromycin. However, I doubt whether cells are just the background or not.
Sharing my troubleshooting with endothelial cells:
1- Starting with less cells increases the ratio virus/cell and therefore more viral particles per cell
2- Try to allow expression of pLKO,-1 puromycin-resistant gene for a minimum of 48 hrs before adding puromycin
3- Your puromycin concentration is relatively high, maybe you can do a titration to lower it down. (in my case I can select endothelial cells in 0.8 ug/ml in 2 days)
4- Whatever cell is left I think it express the puro-gene...I doubt that cells would survive without it in the concentration you are using....
Hope this helps
for siRNA/miRNA transfection of RAW 264.7 cells my colleagues use Viromer BLUE and for plasmid/shRNA transfection they use Viromer RED. By using Viromers they got up to 100% transfection efficiency.
Viromers are a new kind of transfection reagents developed from Lipocalyx (www.lipocalyx.de). They are synthetic polymers zero in charge and very gentle to the cells. Due to their active endosome escape mechanism my colleagues get higher transfection efficiency compared to standard transfection reagents.
Please find attached some data.