"CNS specific knock-outs for cre-lox"

Paul Orban orban at embl-heidelberg.de
Sun Sep 29 08:34:54 EST 1996

In article <960926191314_318231441 at emout12.mail.aol.com>,
Neurobiol at aol.com wrote:

Much deleted, from earlier posters 

> First, I believe there was a report at the mouse meeting of a cre-lox
> knockout made by crossing a cre transgene driven by the nestin promoter into
> a strain that was knocked out for something (I forget what ) that is lethal
> in embryogenesis when homozygous.  I've been too lazy to look up the
> expression pattern of nestin, but I gather from your comments that the PNS
> should have been affected, too.  Does anyone have a better knowledge of this
> work?  I wasn't at the meeting and heard it secondhand...
> Second, Martin, I agree with you that it is very nice but not sufficient to
> restrict the knockout to a particular cell type by breeding to a tissue
> specific promoter-cre mouse line.  I have a couple of reservations.  The
> first is practical.  Not a lot of promoters are really tissue specific, and
> it doesn't take much cre expression to irrevokably alter a cell that might be
> the first in a lineage of any organ.  I suspect there will be a few
> undiagnosed chimeras caused by this method if people don't look at the
> animals thoroughly.   The second reason I think that the cre needs to be
> delivered temporally is of course the issue you bring up-  that delivery of a
> knockout to a specific cell type- by retrovirus? naked DNA? liposomes?- is
> the next step, AFTER the simple knockout is replaced with the
> tissue-restricted knockout.
> Interesting stuff.  I'd like to continue this discussion.  Anyone else?
> Jeanne Loring
> jloring at mdyn.com 

Just to reduce any confusion .. if the mouse meeting you mean was the Cold
Spring Harbor Mouse Genetics meeting, then 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.
This is best done by crossing to a target strain and looking for the
outcome of the Cre-mediated recombination event on a tissue by tissue
basis at least, but ultimately on a cell by cell basis if possible. The
most convenient way of achieving this would be via a "reporter" mouse i.e.
a mouse with lox sites around a single-copy sequence so that the result of
Cre activity is a signal which is easily detectable at the histological
level. Several groups have made reporter mice using the onset of lacZ
expression as the signal of Cre activity, and the most promising of these,
to my knowledge, was 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". I wasn't at the meeting myself so this is only
as reported by a colleague ..... 

As you say, not a lot of promoters really are absolutely tissue specific,
but whatever the method of introducing Cre activity, one has to assay the
result in some way, and recombination events will have to be characterised
in time as well as in space.

Paul Orban

Paul Orban
Differentiation Programme
EMBL, Meyerhofstr. 1
69117 Heidelberg
ph 49 6221 387 425
fax 49 6221 387 516

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