Call for suggestions! (fwd)

malacins malacins at indiana.edu
Wed Jul 2 15:07:37 EST 1997

Wonderful home page, which many uronet readers might want to be aware 
of!  cheers, gmm

George M. Malacinski, Ph.D.
Professor of Biology
Department of Biology
Indiana University
Bloomington, IN  47405
Fax (812) 855-6705

---------- Forwarded message ----------
Date: Tue, 1 Jul 1997 11:33:49 -0400
From: Peggy Kolm <kolm at wi.mit.edu>
To: malacins <malacins at indiana.edu>
Subject: Re: Call for suggestions!

Dear George,
        Our lab has been using the Kroll/Amaya transgenic protocol in
Xenopus.  We have found the helpful hints from Enrique Amaya at :
http://www.welc.cam.ac.uk/~ea3/    to be very useful. In particular, it is
important that the sperm nuclei are not damaged before injection.
Additionally, the Xenopus eggs are NOT activated before the sperm nuclei
are injected. I believe it is the injection process itself that activates
the eggs.  All that is done to the eggs before injection is the removal of
the jelly coats.   It is my understanding that the more quickly the eggs
are dejellied/injected after leaving the frog, the better the results.
A web page with your experiences/results would be useful not only to the
urodele community, but I suspect to us Xenopus people as well.

Peggy Kolm
kolm at wi.mit.edu

>Roy Tassava (Ohio State Univ) and I have decided to have a go at making
>transgenic axolotl embryos, following (more or less) the protocol developed
>for Xenopus.
>We have accomplished the following:
>        Worked out a protocol for electrically activating freshly spawned
>        unfertilized eggs. Once "shocked" the animal hemispere pigmentation,
>        which begins looking like a tiny donut is stamped onto the animal
>        evens out and the donut disappears as pigment blankets the animal pole.
>        Developed a scheme for swelling axolotl sperm heads (a prerequisite,
>        apparently, for successful uptake of a foreign gene).
>        Prepared a marker gene construct (which is functional, for it is
>        expressed when used in cell-culture transfections).
>        Devised a dilution scheme for injecting 1-4 sperm per 20nl
>        Successfully injected large numbers of eggs, with only minor
>        leakage of cytoplasm.
>        Etc.
>HOWEVER, virtually NONE of the eggs (electrically activated)--injected
>with either fresh (crude extract) sperm, swollen head sperm, or
>swollen/construct-treated sperm--cleave!
>What have we done wrong?  What do we need to do to prepare axolotl eggs
>so that they will respond to injected sperm by cleaving?
>We have done various control exercises, including varying the time
>between shedding of the egg and electrical activation, varying the time
>between activation and injection of sperm, varying the concentration of
>sperm injected, etc.  That is, we believe that the obvious "little, easy"
>things we have already checked. Mark Parker--our injection expert (supreme)--
>has injected (during the course of these exercises) literally hundreds of
>eggs with this or that preparation of sperm, buffer control, etc., with
>virtually no success.  Eggs look great the next day: Only  minor leakage;
>pigmentation uniform in the animal hemisphere (presumably, the indicator
>of egg activation); etc.
>I believe we are working on a FALSE assumption: The way to get cleavage
>is to inject sperm directly into electrically activated eggs.
>What is wrong with that assumption?
>Please, somebody, help us get past this roadblock.  Once past it, I
>believe it will be a downhill coast.
>If we succeed, urodele research around the world ratchets up a BIG notch!
>Our goal is to prepare a complete web page, with color photos of each and
>every manipulation, so that the protocols will be in the public domain.
>Thus, everybody from Bloomington to Carracas to Timbukto will gain.
>cheers, gmm
>George M. Malacinski, Ph.D.
>Professor of Biology
>Department of Biology
>Indiana University
>Bloomington, IN  47405
>Fax (812) 855-6705

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