I am trying to design an axolotl transgenic by using a restriction enzyme
mediated insertion of DNA into sperm nuclei. Now the protocol that is
used in Xenopus requires sacrificing the frog and though periodically we do
sacrifice 1 or 2 for tissue, I may need more sperm than what that would give.
So, being the males lay the nice spermatophore cones I want to try to isolate
the nuclei from there. The only problem I am having is getting rid of the
extra gel coat of the cones from the sperm itself. After spinning the tips of
the cones down and several washes etc I can't get rid of the gel. I have not
yet tried a 2.5% cysteine solution that is used to chemically dejelly Xenopus
eggs but will be trying that soon.
I was wondering if you knew of any method to separate the sperm from the jelly
cones they are laid in? I need to remove this before I can demembranate the
sperm to isolate the nuclei.
Thank you for your time and suggestions.
Sincerely,
Matt McLean
MCLEANM at VAX.CS.HSCSYR.EDU
