Finding the location(s) and copy number of stably transfected genes...
jtNOjtSPAM at neurosearch.dk.invalid
Thu Mar 16 05:08:16 EST 2000
You can determine your number of integrations by Southern
blotting. If you find a clone with only one integrant (I assume
that is your goal), you can sequence the regions flanking your
insert with insert-specific primers pointing away from your
If you have multiple integrants, you cannot sequence directly on
you gDNA. Subcloning your insert and flanking regions could be
the way to get around it. FISH on isolated chromosomes might be
If you want to be able to direct integration in a more specific
manner, you could take a look at Invitrogen's Flp-In system.
Briefly, you first make a cell clone with one and only one so-
called FRT-site integrated into the genome. This is done by
conventional transfection, and therefore you have to screen a
number of clones with Southerns. When you have obtained a
suitable clone, you can direct subsequent genomic integrations
to the FRT-site in that clone by the use of a Flp-recombinase.
In theory, you will get integration in the same site every time.
How it works in practice, I am about to find out...
Hope this helps
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