Issues with dCAS9-BFP line

1

I am writing regarding the dCas9 cell line (Cat. No. AICS-0090-391) that we recently purchased from the Coriell Institute. We tested the cells for the BFP tag but were unable to detect any signal using our Cytation5 imager or by flow cytometry. For flow cytometry, we included both a non-tagged negative control and a dTomato-expressing control, but we still did not detect a BFP-positive population (happy to provide flow data/plots if needed). We are concerned that the absence of detectable BFP signal may indicate that the dCas9 protein is not being expressed. We were advised to post on this forum to see if anybody had advice/suggestions.

Thank you,

Marco

2

Hello Marco,

We suspect this is an issue with detection of the BFP signal. It should be expressed but you may just not be seeing it as it is very dim and perhaps the optimization or detection level for your instruments are not set for such a low level of expression. The dim BFP signal does not mean dCAS9 is not expressed though and if you do a knockdown experiment, we would expect it to work even if you are not able to visually detect the BFP. If you include a good positive control you should be able to easily verify the activity of dCAS9 is on and working. Hope this helps you with your experiments!