Lenti viral Vector with GFP - (Apr/13/2009 )
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..
1. It should be fine if the center of the target sites (around 9-11th nt) are different between 2 genes. You might want to design more shRNAs
2. It sounds like your model cells are already "GREEN". If so, clone shRNA oligos to other lenti-shRNA vectors with "drug selection".
Also if the "seed" region of the siRNA doesn't perfectly match the off-target gene, it should be fine.
Do you know why control cells show GFP fluorescence? Have they been infected before or just background fluorescence?
Regarding your second question, keep in mind that some cell types, like some neurons, have some autofluorescence.
THANKS A LOT!! SUGGESTIONS WERE REALLY HELPFUL!!!