4 replies to this topic

[Star*]

Hello!

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..

[Functional Screens]

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".

[pcrman]

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?

[gfischer]

Regarding your second question, keep in mind that some cell types, like some neurons, have some autofluorescence.

[Star*]

THANKS A LOT!! SUGGESTIONS WERE REALLY HELPFUL!!!