oocyte expression cloning

Michael Coady coady at ERE.UMontreal.CA
Wed Mar 8 14:50:40 EST 1995


In article <asch-0703951334260001 at mac102219.med.cornell.edu> asch at mail.med.cornell.edu (Adam S. Asch) writes:
>In preparing pools of transcripts for injection into oocytes, the thought
>occurs to me--why bother?  Does anyone have any experience using DNA
>(rather than RNA transcripts derived from it) for oocyte injections?  Any
>theoretic prohibitions?

	You could delve into your library and check out an article in
FEBS Letts vol. 356, pp 174-178 (1994) by (cough-cough) Coady et al.
We isolated an amino acid transporter (one that a few other groups
had already snagged) by injection of DNA rather than RNA.  Each method
has advantages and disadvantages.  The problems with the DNA injections
include the fact that only a minority of the oocytes that you inject
actually express the DNA that you've injected; this proportion has varied
between 5 and 50% in our hands.  There are a couple of vectors that 
have been used for this purpose.  We're using pMT21, which is a variant
of a vector that was analyzed for this very purpose by Swick et al
in PNAS vol. 89, 1812-1816 (1992).  They showed that coinjection of
the same vector with an alkaline phosphatase cDNA permitted identification
of the oocytes which were expressing the injected DNA.  This is what
we've done but I have to tell you, it's a big pain in the ass.  It's
never worked all that well for us (might be due to the dilution of
SEAP 10 x by the other, desired DNA); it uses a lot of plates and a
lot of tips and a lot of time.  I'm still working on a better way of
doing this but I'm not quite ready to report on it yet.
	So, I hope that this rambling discourse has answered your
questions.  Feel free to email me with more questions if you like.

Mike


-- 
Michael J. Coady
COADY at ERE.UMONTREAL.CA
The opinions expressed above are solely those of the author and do not,
in any way, shape or form, represent the Universite de Montreal.



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