1 reply to this topic

[indu]

Hi guys,
my retroviral constructs some how don't seem to express the genes that are cloned in....these constructs were used by several people and i've procured them from ADDGENE (pMIGs)....first time when i made the prep and did transient transfection in HEKs, they expressed the GFP reporter (under IRES) and later on, for some reasons, absolutely nil or low GFP expression.....since am struck up at the packaging cell transfection stage (low transfection efficiency), i couldn't proceed with the viral prep at all....don't know  whether the IRES is the problem....can any one see any basic problem here.....am almost frustrated with this problem....would be happy to have any suggestions please. IM.

[NemomeN007]

indu, on 09 August 2010 - 02:32 AM, said:

Hi guys,
my retroviral constructs some how don't seem to express the genes that are cloned in....these constructs were used by several people and i've procured them from ADDGENE (pMIGs)....first time when i made the prep and did transient transfection in HEKs, they expressed the GFP reporter (under IRES) and later on, for some reasons, absolutely nil or low GFP expression.....since am struck up at the packaging cell transfection stage (low transfection efficiency), i couldn't proceed with the viral prep at all....don't know  whether the IRES is the problem....can any one see any basic problem here.....am almost frustrated with this problem....would be happy to have any suggestions please. IM.

Have you tried using new 293T cells?  sounds more like bad cells than a plasmid issue.