transgene insertion and repetitive sequences

[Ed Siefker]

Tom Anderson wrote:

> 
> Once you've got that far, order up a genomic clone (or several) from
> normal mice covering the area of the insert and use it as a probe on a
> Southern blot. Comparing the pattern to that from normal mice will tell
> you with, i think, a high degree of precision where your insert is.
> 
I'm not sure I understand this.  We do know that the transgene is in
band 3A3.  If I get a BAC from this region, how would I use it as a
probe?  It's kind of big for that isn't it?


> How about doing this protocol, but using an enzyme which cuts inside the X
> chromosome repeats? You wouldn't get your transgene in the clones, but you
> could get the X-3 junction, and the clone would be smaller. You'd need to
> use a primer matching the repeats for the screening step. You run the risk
> of just cloning repeats, but if you gel-purify the digest to remove all
> the fragments smaller than the size of a repeat, you avoid that.
> Alternatively, double-digest with an enzyme which cuts the repeats and one
> which doesn't (which give different sticky ends), and clone into a vector
> cut so it will only take inserts with one end from each enzyme.

Hmmm. Those are good ideas, I hadn't thought about gel purifying the 
digest.  I should be able to get rid of the endogenous X chromosome 
sequence that way too.


> 
> Another way of getting rid of real X DNA would be to find some genomics
> people and get them to flow-sort chromosomes from your mice. Once people
> have things set up for doing this, it works very well.
> 

Hey neat, I didn't know they could do that.

> Aha! Do FISH-and-Southern mapping, then make Boulter clones with an enzyme
> that cuts the X repeats, then do the screening step with a primer that
> matches the closest known bit of chromosome 3 sequence. This does require
> that you map the location to within a few kb, though.

Yeah, I'll have to ask the people who did our FISH if they can get us 
any finer mapping. Thanks a lot for the ideas.
-Ed Siefker