Hi Enes,

I think it this is a problem of efficiency.

I have no experience with your cell line but I haver never seen a problem of transduction linked with the number of passages. But I already seen epigenetic interferences, IRES problems.....

I think the design of your vector could be optimized to otain better results.

Let me know if you need further information.

Regards

Nicolas

Dear Nicolas,

Thanks for your kindly response.

In this situtation, even IRES cause the problem, it should not effect my efficiency control due to the fact that GFP is before IRES so maybe NeoR expression can be reduced.

To be clear , I update my question in a better way, Does the expression driven by CMV promoter can be reduced in higher passage? and the second question is , Does the transfection efficiency can be reduced by passage numer?

Hi Enes

1.

CMV is sensitive to epigenetic regulations with time (and so with the passages even this is not exactly this fact which is responsable) you can observe a diminution up to extinction of your gene drived by CMV. EF1 is a weaker promoter than CMV but less sentitive to epigenetique regulations.

2.

In my experience passage doesn't reduce the transduction efficiency.

Regards

Nicolas