p-ashby at nimr.mrc.ac.uk
Wed Mar 12 12:42:50 EST 1997
In Article <5g6ekm$kqv at mserv1.dl.ac.uk>, "Joseph Cheer _ Coordinador de
Neurobiologia. FINC _" <cheer at javercol.javeriana.edu.co> wrote:
There are a lot of people in the newsgroup who are not sirs.
>I am a Biologist who will start a PhD in Molecular Neuropharmacology
>this fall. I am very interested in the use of transgenic mice (specially the
>use of embryonic stem cells as a vehicle to mutate specific genes). I do
>have some protocols but I have one question that seems to remain
>unanswered. After pro nuclear microinjection how does the DNA template
>integrate the host genome. Is it by homologous recombination? I would
>like to know what the exact mechanism is. Could please shed some light in
>this subject for me?
DNA introduced by pronuclear injection does not integrate by homologous
recombination otherwise a lot of sequences used would not integrate and
those that did would lead to knockouts. If you could make knockouts with
pronuclear injection I would have a stack of Nature papers by now :-)
Transgenic constructs are generally inserted into one or more places in the
genome in a semi-random fashion. They are in tandem head to tail arrays of
from one to dozens of copies, although one copy only can be very hard to
get. I don't know for sure but I think they are usually blunted into place.
Disruptions caused by transgene insertions are usually because the transgene
has randomly inserted into coding sequence.
Hope this helps.
Peter Ashby National Institute for Medical Research
Eukaryotic Molecular Genetics London, England
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