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Trouble about overexpression by lentiviral transduction - Why no protein of interest expressed!? (Mar/20/2010 )

Hi! Everyone!
I uesd the pLKO AS2 as my lentiviral vector. Visit My Website
I inserted 750-bp genes of interest into the vecors. The processes of lentiviral production and infection seem successful because my infected cells (LNCaP, a prostate cancer cell line) survive well after G418 selection(about 3 days). However, no exogenous protein was expressed when I checked the expression level with Western blot!
It seems that my genes in constructs have frame shifts. But it is little possible because all of 4 constructs have the same failure results! Could it happen even when I used Pfu polymerases to amplifiy my genes?! I inspected my primers and they are right! Anyway, I will sequence all the constructs again!
This awesome troulble almost killed me! Plz would anyone who has done this technique give me a little suggestions? Thanks!


If you have frame shifts then your protein won't translate properly and it won't express! :lol:

You're on the right track to go back and check all of your sequencing, chances are they all have the same frameshift problem. If this is the case, a new clone will need to be made before you attempt any expression. Find the frameshift problem and correct it before doing anything else. It's always a bummer to find out at the expression stage, but makes us more careful to verify the correct sequence the next time.