Oligo primers for shRNA construction - (Mar/14/2012 )

Hi there

Please forgive my silly question. I would really like to understand this.

I need to construct a shRNA cell line and the first step, from my understanding, is designing primers to knock down my gene of interest.
Why do I need primers for constructing shRNA? I am assuming that I need to run a PCR reaction with it but what the purpose is, aside from the obvious amplification, is something I dont understand since I do not have template. Since there is no template, how is the oligo used to knock down the specific gene? I will be using lentiviral vector for construction

Seems that no one likes to share information in my lab so i've been searching the net and figuring bits and pieces out myself but running into a few basic stumbling blocks.

Thank you so much for any help!

Hi,

I have never used any silencing technique but I am also trying to understand these things. More specifically I was interested in antisense oligonucleotides. Anyways while reading I came across two really good links that helped me to understand, hopefully it will help you as well. Here are the links-
http://www.scbt.com/gene_silencers.html

http://www.genomeweb.com/node/941787

amj2