Need expertise on DNA recombination mechanisms
blb0 at musicb.mcgill.ca
Tue Apr 28 10:27:32 EST 1998
Here's a challenge for someone who knows about DNA recombination mechanisms:
I have been working for the last little while on generating mouse
embryonic stem cells with both copies of "my nightmare gene" (MNG)
targeted. These clones are to be used in in vitro differentiation
experiments. To do this, I have built two targeting vectors by using two
different drug resistance genes namely, neomycin and hygromycin. Both
vectors are built from the same 10-kb fragment containing a 2.5-kb exon
bordered by intron sequence.
So, first I generated the neo vector by replacing a small portion of
coding sequence in the exon (50-bp) with the pMC-neo1-polyA sold by
Stratagene. When I screened my clones, one third of the G418-resistant
clones turned out to be a positive recombinant...an astounding rate by any
Second, I made my hygromycin-resistance vector. It's based on the same
10-kb fragment except for the following: the hygro is in the opposite
orientation to MNG and, due to restriction site constraints, I took out
0.6-kb (instead of just 50-bp). When I screened my clones this time I was
not quite as successful: Total recombinants = 0
At first, since MNG is expressed at low levels in ES cells, I thought
it might be implicated in very early development, so much so that the
survival of ES cells in vitro may be compromized. To verify this, I redid
the electroporation using wild type ES cells and the hygro vector: I got
1% of recombinants this time (2:200). When I electroporated this
heterozygous clone with the neo vector, I got the same recombination
efficiency as when I electroporate wild type ES cells with the neo vector.
So now I know this:
1. MNG-null cells can prolifereate in culture.
2. Either the hygro gene itself prevents recombination or, more likely
I think, something in the portion I took out is a recombination hotspot.
According to these results, the presence of this hotspot would increase
the recombination rate by 3300% !
Now, I have sequenced this DNA segment but now I'm not sure exactly
what to look for.
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