2 replies to this topic

[cathyolive]

Hello all,

I am working on non-viral vector DNA and trying to figure out why the transgene is silenced in our vectors.  To that end, I'm trying to come up with a cell culture model.

I was wondering if anyone's had a stab at doing ChIP on non-integrated, episomal forms of plasmid DNA?

Do you start with the Hirt method to isolate your extrachromosomal DNA first and then proceed to ChIP as 'standard' (haven't picked a particular protocol yet)?

Would this pDNA be much more susceptible to sonication?  (I've read the fab article on here by Dukey about sonication vs MNase digestion- too bad MNase digestion still requires sonication though).

Many thanks in advance for your help!

Edited by cathyolive, 08 December 2011 - 04:00 AM.

[chabraha]

Someone a few labs down the hall tried ChIP on a plasmid trying to confirm pol III binding to promoters and found it very tedious..........I think we both came to the conclusion that during transfection you end up with a ridiculous amount of DNA into the cell..........some of which is not functional................and creates very low % Inputs, making meaningful comparisons to cellular controls very difficult..........you could try and answer your sonication question by southern blotting for your plasmid to check for shearing efficiency. If your transgene is a transcriptional repressor you may be experiencing some sort of feedback...........but that's totally a shot in the dark.

[cathyolive]

Thanks for your reply, much appreciated!