No reporter expression

[Crispy Fry]

We edited a transcriptionally silent gene by inserting a cassette with both a reporter and an antibiotic resistance marker. Integration is confirmed, and selection worked fine. But during differentiation, the reporter doesn't show any expression. Could the resistance gene be interfering with reporter expression, or is it more likely tied to the silent nature of the locus?

 

[DrRespectful]

I think it's unlikely the resistance gene is blocking reporter expression, more likely, the silent state of the gene's locus is keeping the whole cassette off during differentiation. Since the gene is naturally inactive, the reporter wont turn on unless the surrounding chromatin becomes active

 

[Caitlyn P]

We edited a transcriptionally silent gene by inserting a cassette with both a reporter and an antibiotic resistance marker. Integration is confirmed, and selection worked fine. But during differentiation, the reporter doesn't show any expression. Could the resistance gene be interfering with reporter expression, or is it more likely tied to the silent nature of the locus?

If your cassette landed in a region with tight chromatin or repressive histone mark, it's more likely tied to the silent nature of the locus, even with selection working, transcriptional silencing can still suppress your reporter, try adding an insulator element or repositioning the reporter with its own strong promoter to see if that helps

 

[Saywha]

The resistance marker could be interfering, but only if you have transcriptional interference, otherwise I'm with @Caitlyn P and @DrRespectful on this. YOu can do a test for chromatin accessibility to see if the locus remains repressive

 

[crisprnavigator25]

Okay, that makes sense. Checking how open the chromatin is should tell us if that spot is still shut down.