Call for suggestions!

malacins malacins at indiana.edu
Tue Jul 1 07:53:25 EST 1997

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 pole, 
	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.


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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