Have you heard great knock down with GIPZ transfected into cells but after several passages, knockdown is lost?
We have heard of failure of integration with transfections. You can increase the rate of integration by linearizing the plasmid prior to transfection. For pGIPZ, FspI cuts at base 10,755 (inside amplicillin marker). There are a couple of reasons that knockdown could be lost over time. The promoter can become methylated and therefore silenced. If that is the case, then puromycin resistance and GFP expression will also decrease over time. There is also evidence to suggest that transfections of GIPZ (which contains the SV origin of replication) in cell lines that do not contain the large T antigen will eventually be lost because the large T antigen is required for replication. In this case the plasmid may still be present in the cells but is not replicating. Cells should be kept on puromycin to ensure that only cells with the plasmid continue to survive. Another thing to consider is the possibility of a feedback loop. In some cases, loss of a gene will initiate a feedback loop that will compensate for the knockdown by increasing the overall expression of the gene.