RE In vitro recombination

Andrei Popov ANDREI.POPOV at bbsrc.ac.uk
Sun Nov 12 07:30:38 EST 1995


In Article <483cft$57r at mserv1.dl.ac.uk>, Andrei Popov
<ANDREI.POPOV at bbsrc.ac.uk> wrote:
>>Does anyone know if in B-cell differentition,  the D-J recombination and
>>the Variable Heavy-DJ recombination reactions have been successfully
>>performed in-vitro.  A friend of mine said that he thought Dr. Stuart
>>Kauffman of the Santa Fe Institute has done this, but I haven't been
>>able to find any references on this.  Any help would be greatly
>>appreciated.
>
>No. It has not been done.
>M.Sadofsky (spell?) and coauthers published
>a couple of months ago in Cell a paper on V(D)J recombination
>in vitro but.... it was very far away from total success.
>
>

>Well, actually I think that it a little closer than no.
>M Gehlert's (?) group did have a paper on it, and I think that
>it was pretty good, using cell extracts.  He presented new
>data during the 9th internation congress on immunology showing
>that placing recombinant rag I and rag II in a hepes/mops buffer
>with a radiolabled RSS 50mer oligo gave a cut product.  This isnt
>exactly recombination, but shows that the rag s are directly
>involved in recombination, and bona fide invitro rec shouldnt be
>far off.  also, the test for activity of the rag s has been using a
>plasmid with rss's in 3t3s with transfected rag i  and ii.
>this seems to work pretty well.   and i wouldnt really call it
>invivo.
>


>Regards, Ralph
>
>
>
>Ralph M. Bernstein
>Dept of Micro/Immuno
>University of Arizona
>Ph: 602 626 2585
>Fx: 602 626 2100
>url: http://lamprey.medmicro.arizona.edu
>     http://radon.gas.uug.arizona.edu/~bernster          

OK. I've got the paper: Initiation of V(D)J recombination in a Cell-Free
System. Cell, v81, 925-34.
Only the first step of the V(D)J recombination (formation of the hairpin
structure after double-strand break, DSB) was achieved. In all
these experiments RAG-1 and RAG-2 products could only enhance the activity
of the nuclear extracts from pre-B cells. I can hardly call this
a defined recipee for in vitro V(D)J recombination in the tube.

Regards

Andrei Popov

                              




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