Flp-recombinase target site integration in 293 T-REx cells - (Aug/15/2011 )

Hi everybody,

I have a question concerning the 293 T-REx cells line that Invitrogen offers for the use with their Flp-In T-REx kit for stable integration of a gene of interest in a single genomic site.

I have used this system to insert a binding sequence for a protein of interest into the genome. This is supposed to serve as a positive control in a ChIP-seq experiment to figure out if and how many off-site binding sites my protein has in the human genome. At this point I am using the sequence of the inserted plasmid containing the known binding site as a "mini-chromosome" in addition to the hg19 reference genome for alignment and peak calling. However, it would be preferable to know where the FRT site in the original 293 T-REx genome located is.

There is no information about this anywhere to be found in the manual (at least I didn't find it). Possibly the easiest way to figure out the location of the site would be a junction PCR (e.g. LAM-PCR). I thought it might be worth it asking first here if anybody knows already the answer to that question. Has anybody that's working with this cell line bothered to see where the insertion site is by any chance?

Thanks for any help,

Wonko

I doubt that anyone has actually looked to see where the insertion site is as it doesn't really matter for most things, unless it disrupts a critical gene sequence.