Lenti viral Vector with GFP
Posted 13 April 2009 - 05:22 PM
I'm very new to this subject but I'm trying to knock down certain genes in mammalian cells using shRNA. Therefore, I've several questions...which may sound very basic... sorry for that...
1. I designed a shRNA for a known gene using one of the company's website but when I blasted the sequence on NCBI blast it showed 100% similarity with the gene I wanted to knock down and 90% similarity with some other gene... What should be the least similarity of ShRNA sequence to knock down certain gene?
2. I'm trying with lentiviral system with GFP but I do not have good protocol to confirm the transduction.. coz I'm being able to see gree colours in control cells (cells with out viral transduction). Do I need to fix the cells before observing or what should I do?
Please help me out..
Posted 13 April 2009 - 06:28 PM
2. It sounds like your model cells are already "GREEN". If so, clone shRNA oligos to other lenti-shRNA vectors with "drug selection".
Posted 13 April 2009 - 08:14 PM
Do you know why control cells show GFP fluorescence? Have they been infected before or just background fluorescence?
Posted 15 April 2009 - 11:31 AM
then heaven will be yours, before you meet your end