Login

Join for Free!
117190 members


Can you recommend a vector?

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

Can you recommend a vector?

Postby JimmyTee » Wed Jun 15, 2011 3:17 pm

Hi all,

I am doing some mutation analysis. I have generated 4 mutants and a wild type and have been trying to transfect my cells and generate single cell stable clones. The problem is that the gene is under the control of the CMV promoter and we are getting constitutive silencing, this even happens when using a GFP reporter, where there is no phenotype. We can maintain expression (as detected by western and qPCR) of the transgene for 8-12 cell passages, but then the CMV becomes silenced. We are working in Raw 267.4 cells, a macrophage line which are apparently notorious for this.

So far i am looking for a human promoter within the plasmid which is inducible by the addition of a drug or something similar.

If anyone has any experience of this or has a suggestion for a plasmid into which i can clone my mutants then it would be great to hear from you.

Thanks all,

Jimmy
JimmyTee
Garter
Garter
 
Posts: 3
Joined: Mon Mar 14, 2011 10:34 am

Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 2 guests

cron