knockdown: yes; GFP: no ?! what is this? - (Apr/07/2011 )

Hi guys,

I have cell lines trasduced (retroviral transduction) with constructs containing shRNA's which give knockdown of a certain protein. The constructs also contained GFP-IRES and puro res. gene. After transduction I selected by adding puro.
The cell lines are OK. In one the protein is knocked down on mRNA and on protein level. Other cell lines are less succesfull. So I checked for GFP and to my surprise the cells are not green under fluorescence microscope and not even in the FACS! Including the cell line which has succesful knockdown!!
Does anyone have an explanation for this or similar experience?

Love to get any comments.

Ytje

what is the GFP-IRES (internal ribosomal entry site) linked too?

It is part of the LZRSpBMN vector.
I used it for the expression of a small secretory protein.