Hello everyone. I am interested in inserting a large-sized fragment (~20-30kb) into the genome of mammalian cells. What methods can I employ to achieve this? Additionally, how can I obtain the plasmid containing this fragment? Both random insertion and locus-specific insertion are acceptable to me, but I prefer the latter.
The easiest solution would be to use a serine/threonine recombinase like CRE or FLP to integrate your plasmid into the genome. If you want a specific insertion site use CRISPR to make a double strand break and then insert a landing site with selectable markers between the two homology arms. With recombination sites at either end of the selection cassette you can then do cassette exchange. Using a lethal gene driven by a tet operated promoter can allow for selection of your fragment being inserted into the genome.
For getting your fragment I advise checking if there is a cosmid library with your region of interest. Else you may need to clone in fragments and then do a Gibson assembly.
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