Protocol for delivering shRNA through lentivirus - Please! - (Apr/04/2008 )

Hi all,

Very soon I will be starting an altogether new experiment. I need to knock down the expression of certain proteins in some of the cancer cells using lentivirus. Can anyone please throw some light on the issue and also give me some protocol on how to go about doing that. Thank you.

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We've had good success with the Invitrogen Block-iT kit. Excellent shRNA expression and knockdown, plus it's available in a lenti. An added bonus is that the shRNAs are expressed co-cistronic with GFP, so you can directly relate GFP expression to shRNA expression. There's lots of info on the product webpage:

http://www.invitrogen.com/content.cfm?pageid=11280

We also used the free invitrogen shRNA design tools to pick candidate shRNA sequences, and of the 8 we tested, four gave excellent knockdown (>90%) and three of the others were >70%.

Ginger

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thanks a lot, that was a great suggestion.. will let you know how it came out.............

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Hallo consider gene silencing techniques as siRNA, it specifically cleave mRNA and thus block translation of the target protein.Eficiency is around 90% of expresion is decreased
good luck

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Thanks a lot for the suggestion Baxapoptoaia!!!!!

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The slight problem here is that if you need a lenti, by definition you can't use siRNA. There is a big difference between directly adding double-stranded siRNA molecules to the cell using e.g. lipofectamine, and using a viral (i.e. DNA-based) delivery method, which requires that you use hairpin-based miRNA or shRNA.

Ginger

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