I'm trying to package pBabe-KRAS, pBabe-beta-catenin and pBabe-PIK3CA retroviral constructs. So far, I have no luck with beta-catenin or PIK3CA although there is no issues with KRAS or empty vector. I have tried different packaging plasmids (pUMVC, VSV-g), (pUMVC, pMDG.2), (pCL-Eco). I have sequenced the plasmids and there are no issues? I have used different transfection reagents? I have even subcloned both genes on pMSCV backbone and faced the same issue? These plasmids have been used in other publications and I have used the exact protocols and did not work? Am I missing something?
Thanks,
Abdalla
Please post the publication(s), where the plasmids were used! What is your result, how do you titer your virus? Are the transgenes expressed in the producer cells from your pBabe-vectors?
I got both plasmids from Addgene
https://www.addgene.org/24204/ (beta-CATENIN)
https://www.addgene.org/12525/ (PIK3CA)
I normally don't titer the viruses as I always get good transfection efficiency with the plasmids I'm using. The transgenes are expressed in the producer cells from pBabe vectors as they are resistant to puromycin. Please let me know if you require further info.
Thanks,
Abdalla
I would strongly recommend to check whether your producer cells release any virus at all by isolating viral RNA from the supernatant and quantify via RT-qPCR. Maybe the virus is released, but (nearly) non-infectious for whatever reason?
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