"CNS specific knock-outs for cre-lox"

Neurobiol at aol.com Neurobiol at aol.com
Sun Sep 29 14:12:31 EST 1996


Subj:   Re: "CNS specific knock-outs for cre-lox"
Date:  Sun, Sep 29, 1996 8:27 AM PDT
Subj:   Re: "CNS specific knock-outs for cre-lox"
Re:  orban at embl-heidelberg.de (Paul Orban) message
In response to my questions (text deleted)  Paul wrote about the discussion
of the cre-lox knockouts now being reported at meetings:
Paul's message:
>... the CNS Cre-Lox knockout in
>question was of ErbB4, and it was indeed a Nestin-Cre mouse ....  (I made
>the  construct for the Cre mouse). We don't yet know about PNS effects of
>this knockout, but we have indeed seen Cre expression in the PNS in these
>mice. Whether peripheral or even unexpected sites of Cre expression MATTER
>in a particular case will of course depend on the lox-flanked target and
>where it is expressed.
>IN the "early days" of the use of the Cre/Lox system in mice, it was
>unkown how much Cre expression would be sufficient to achieve the desired
>event in vivo, so several workers (myself included) did design transgene
>constructs to drive Cre expression. Since then, some labs have made/are
>making Cre knock-ins to maximise the use of endogenous context to direct
>Cre expression ..... whether or to what extent this will overcome the
>problem of the leakiness alluded to in this discussion is not yet known.
>In either case it is a matter of characterising the Cre mouse carefully.
>....recombination events will have to be characterised
>in time as well as in space.
>.....one also reported at the CSH meeting by Stephen
>O'Gorman. He and colleagues knocked a lacZ construct into the RNA Pol II
>locus with a lox-flanked transcription stop cassette between promoter and
>lacZ coding sequence. He showed whole mount staining from a mouse in which
>Cre activity had occurred at the oocyte stage - and at this level at least
>it was "completely blue". 

Thanks, Paul.  Your mouse was indeed the one I'd heard of.  
The O'Gorman mouse sounded pretty interesting, too, as a technique testing
device.  I heard that Ramirez-Solis also has a system developed for
monitoring cre activity.  Using a knock in of cre instead of a
tissue-specific mouse promoter  transgenic is certainly a "prettier"
approach.  But I've yet to see a side by side comparison of the two methods,
and I'm not sure it's worth the effort to knock in the cre.  I think that
YAC-based transgenes might make the cre delivery more controllable and put
the mouse together more quickly.  But this is a personal bias of mine since
all I know how to do is knock-outs and ins and YAC transgenics.
I'm curious about delivery of Cre via other methods- targeting a tissue has
been an interest of mine for a long time.  You're right of course in saying
that it doesn't matter if a few cells around the body have a gene deleted, if
the main target is hit hard.  The point I was trying to make was that not
everyone will think to look for confounding factors, and it would be very
nice if every cre transgene, whether it is a knock in or an exogenous
transgene, would be accompanied by a cre-detector mouse background-then if a
cell is blue it's lacking the gene (or at least ONE copy of the gene if the
lox sites are in both copies).  It would be best to use a non-lethal knockout
phenotype for this- like CFTR, so you could use homozygous lox-flanked
animals for breeding.
Back to targeting a tissue by other means- have any gene knockout people yet
gotten together with gene therapy people to use a lox reporter mouse to see
if, when, and where the exogenous genes are delivered?
Any reason why this wouldn't work?
Cheers,  
Jeanne Loring



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