We are investigating the function of a gene supposedly involved in cell division. We designed an SH RNA for this gene and wanted to study silencing the gene by cloning it in MU6 vector and transfection into mouse testis TM3 cells. We first cloned the transcript of our intereest in the MU6 vector upstream (5') of the GFP. When we transfected this construct into TM3 cells and grew teh cells, strangely we find that the culture plate developed faint GFP color all over, while very few cells (1-2) only show the fluorescence and loose the same very soon (in a matter of minutes).

Regarding the loss of the background fluorescence over several minutes: if the cells were illuminated under bright light for that duration (e.g. sitting on a fluorescence scope under excitation light) then the loss of fluorescence signal is likely to be due to photobleaching of the fluorochrome.

Regards,

- Jon

Jon D. Moulton, Ph.D.
Gene Tools, LLC
www.gene-tools.com

You might want to look at the cells under low exposure times - and i would strongly recommnd using the "spinning disc" systems from Olympus, Leica or equivalent. The other thing to do, if I have read your experiment setup right, would be to do a flow cytometric analysis with your control and experimental shRNA transfected cells, and look for differences. Since a flow cytometer individually analyses each cell, it eliminates environmental variables such as plate/materials induced fluorescence.