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retroviral transduction problem

retroviral transduction

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#1 caseykay

caseykay

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Posted 17 April 2013 - 04:11 PM

Hi, I am here for help!
I am trying to generate stable cell lines using retroviral transduction. I use retroviral vector MSCV-N-flag-ha-IRES-puro. My genes of interests are 5 homologous proteins.
When I transient transfect these constructs into 293T cells, they all show robust protein expression.
Then I co-transfect the retroviral vectors and pVSV-g (clontech) to packaging cell line gp2-293 cells (clontech, stably expressing gag-pol) to package virus. After the virus collection, I harvest the package cell lines and immunoblot for the protein expression.
What troubles me is that 4 package cell lines show very little protein expression, but 1 cell line has robust viral protein expression. Only virus collected from this cell line is able to successfully transduce my target cells, keratinocytes.
My goal is to generate stable keratinocyte cell lines that have similar protein expression level for these 5 proteins. However, I can only have 1 construct out of 5 works. I have tried several times, and this particular constructs always work but not others.
I am very confused and could someone with experience help me with some tips to resolve the problem?
Thank you very much!
-caseykay




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