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Help! No infection with modified vector!

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#1 litos



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Posted 09 October 2009 - 05:04 AM


It's me again, the retroviral vector guy :blink:

I am having a lot of trouble trying to create an improved retroviral vector based on an previous one from my lab. The vector, based on MoMLV, lies in a cloning plasmid which has a modified 5' LTR with the CMV promoter instead of the U3 (supposedly to increase the viral titer).

To improve transgene expression, we decided to change several things. First, we deleted the majority of the 3' LTR U3 to create a SIN (Self-inactivating vector). Then, we introduced a strong internal promoter beside the transgene, for improved expression. We cotransfected the new vector with our standard helper plasmids in a packaging cell line and everything seemed just fine. Then, we collected the supernatant and ultracentrifugued it to concentrate the viral stock. But, when we performed an infection on our target cells, there were no traces of the reporter (eGFP), we had no infection.

I am very upset, because I have carefully analyzed the cloning strategy and frankly, I do not know where the problem could be. My only guess is promoter interference between the CMV and the strong internal promoter, that leads to a reduced viral titer but... ¿to the extent of seeing no infection? And I do know that U3 regions are tricky and contain some signals that may contribute to correct viral genomic processing and for that reason I have made a deletion which has already been done by others ...

Any thoughts will be more than welcomed.

Thanks a lot.

Edited by litos, 09 October 2009 - 06:25 AM.

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