In Article <5g6ekm$kqv at mserv1.dl.ac.uk>, "Joseph Cheer _ Coordinador de
Neurobiologia. FINC _" <cheer at javercol.javeriana.edu.co> wrote:
>Dear sirs,
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
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Peter Ashby National Institute for Medical Research
Eukaryotic Molecular Genetics London, England
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