Transgenic reporter constructs
bernard at elsie.nci.nih.gov
Mon Aug 26 02:46:48 EST 1996
In article <Pine.SOL.3.91.960825135945.29240C-100000 at lonestar.jpl.utsa.edu>,
spenadeo at lonestar.jpl.utsa.edu says...
>I want to make a construct containing a reporter gene for microinjection
>and production of transgenic mice. Is there any specification in the
>type of vector I need to to use? Can I use a linearized plasmid reporter
>construct that I know works in mammalian cells ?
>Sandra Pena de Ortiz
>University of Texas, San Antonio
I've never seen solid guidelines for this so I'll be reading replies
to this post with a great deal of interest.
Our habit here is to excise the expression cassette (promoter,
cDNA and polyA signal) from the plasmid and get rid of most of the
backbone. My personal practice has been to leave some non-cassette
sequence on either end to "compensate for losses" (no rational
reason but I assume that nucleases within the embryo will have a go at
foreign DNA and there may be some loss during integration). I usually
leave 100 - 200 bp that is free from anything obviously nasty (eg. neo).
Despite this I have found some lines in which I have lost some sequence
from one end or the other - however, my transgene is somewhat toxic so I
could be selecting for mice in which it is inactive/less active.
I have heard that other groups cut the cassettes out cleanly
with no vector sequence at all and still generate mice succesfully.
I have yet to see a side-by-side comparison of preparations with the
same cassette but maybe someone somewhere has done this.
...and no, we've never tried linearised plasmid.
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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