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[icome]

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!

Edited by icome, 20 March 2010 - 01:46 AM.

[Denny]

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

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.

Edited by Denny, 21 March 2010 - 05:04 AM.