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Lentivirus transfection using RAW 264.7 cells - (Jun/02/2014 )

Dear everybody:

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>

Results
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.

-Tom Chang-

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
 
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-ostiaziocan-