[Xenopus] high-throughput devitellinizing?

Sun Nov 1 21:31:35 EST 2020

Hi Levin and Xenopus people:

To hear from you, among them are my very old friends such as Enrique, Richard, Peter, and so on, is extremely nice to me. Basically, I usually have nothing to add to these mails talking about the most updated topics.  This is mainly because I do not have laboratory and do not work any more about Xenopus molecular biology in the last 6 years, and only thing I do is to do some lectures once or twice a week in local vocational schools in Fukuoka, and just send occasionally Xenopus-related articles to newspapers such as the one attached herein as a specimen (This is not attached, because it is too big).  

Today, however, I found that I would like to say just a few words in relation to the statement by Dr. Michael Levin, that "The most time consuming part is removing the vitelline membranes. .......Yes!! Speaking of which: does anyone have an enzymatic method for this yet?  Maybe recombinant hatching enzyme cocktail or something?  Is there a decent way to devitellinize tons of embryos at once?"

Naturally, of course, I have never actually seen tons of embryos before hatching in my previous laboratories in Kyushu Uinversity, in University of Tokyo, and in Teikyo University.  However, when I worked with EDTA-dissociated embryonic cells while I was in Doctor Course student in Kyushu University in 1967 (Shiokawa, Nada, and Yamana, Nature, 213, 1027-1028: Shiokawa and Yamana, Dev. Biol., 16, 368-388), I dejellied thousands of blastulae using 2.5% sodium thioglycolate, pH 8.5 in about 3-5 min, and then tried to dissolve vitelline membranes similarly quickly.  I used various agents and struggled to solve the problem, with no good results.  Of course I tried to use the same 2.5% sodium thioglycolate pH 8.5 to eliminate vitelline membranes with or without trypsin at several different pH.  The only thing that I could get finally was the "finding" that vitelline membranes disappeared after about 5 hours of continued culture in sodium thioglycolate pH 8.5 without trypsin in gentle shaking conditions.  

After that I always eliminated vitelline membranes manually using a very sharped forceps.  Actually, my object was to get a nice dissociated cell culture to be used for labeling experiments.

However, one time I was so irritated, that I sucked blastulae immersed in a dissociating medium (a medium with 0.05 M EDTA) relatively strongly using a pipette with a relatively small opening.  Of course, embryos were disrupted.  However, I found many cells remained intact in the dissociating medium.  Since I saw that, I decided to cut say hundreds of blastulae and neurulae using ophthalmologist's cutting forceps in the dissociating medium, and after gently exchanging the medium into the complete Stearns' culture medium, I used to get a population of nice dissociated cells at each developmental stages.  In embryos at cleavage stage, however, this cutting methods were of course no good.  So, there, I used again very sharpened forceps and got devitellinated embryos just manually.

Lastly I would like to thank Dr. Michael Levin, because his writing reminded me of these very very old story when I was quite young.

Koichiro Shiokawa  

-----Original Message-----
From: xenopus-bounces at lists.mbl.edu <xenopus-bounces at lists.mbl.edu> On Behalf Of Levin, Michael
Sent: Sunday, November 1, 2020 1:04 AM
To: Enrique Amaya <Enrique.Amaya at manchester.ac.uk>; Jeremy Green <jeremy.green at kcl.ac.uk>
Cc: Leon Peshkin <peshkin at gmail.com>; xenopus at lists.mbl.edu
Subject: Re: [Xenopus] high-throughput devitellinizing?

Hi everyone,

From: Enrique Amaya <Enrique.Amaya at manchester.ac.uk>
> The most time consuming part is removing the vitelline membranes

	Yes!! Speaking of which: does anyone have an enzymatic method for this yet?  Maybe recombinant hatching enzyme cocktail or something? Is there a decent way to devitellinize tons of embryos at once?

Best,

Mike Levin

---
Dr. Michael Levin
Vannevar Bush Chair,
Biology Department, and
Director, Allen Discovery Center at Tufts and Tufts Center for Regenerative and Developmental Biology Tufts University Suite 4600, 200 Boston Ave.
Medford, MA 02155-4243
Tel. (617) 627-6161
email: michael.levin at tufts.edu
http://www.drmichaellevin.org/
http://www.cellregeneration.org/
twitter: @drmichaellevin

Center Administrator:
William F. Baga
Tel. (617) 627-5735
Fax: (617) 627-5035
email: william.baga at tufts.edu

-----Original Message-----
From: <xenopus-bounces at lists.mbl.edu> on behalf of Enrique Amaya <Enrique.Amaya at manchester.ac.uk>
Date: Thursday, October 29, 2020 at 7:45 AM
To: Jeremy Green <jeremy.green at kcl.ac.uk>
Cc: Leon Peshkin <peshkin at gmail.com>, "xenopus at lists.mbl.edu" <xenopus at lists.mbl.edu>
Subject: Re: [Xenopus] MISC

    Dear Leon,
    The most time consuming part is removing the vitelline membranes. When I was animal cap king decades ago my preferred method was to use an eye brow hair knife and put the de-vitellinised blastula embryos on their side and decapitate them with the eye brow knife… a very rapid method. If working with another, I would suggest having someone removing the vitelline membranes and another person performing the decapitations, to ensure consistency in animal cap removals. One can easily get through hundreds this way. How many do you need?
    Enrique

    ——————————————————————————————
    Professor Enrique Amaya
    Division of Cell Matrix Biology & Regenerative Medicine
    Michael Smith Building
    School of Biological Sciences
    Faculty of Biology, Medicine and Health
    University of Manchester
    Oxford Road
    Manchester  M13 9PT
    United Kingdom
    Office: +44 161 275 1716
    Twitter: @Enrique1Amaya

    On 29 Oct 2020, at 11:08, Green, Jeremy <jeremy.green at kcl.ac.uk> wrote:

    Dear Leon, 
    With a bit of practice and well-sharpened Dumont #5 forceps, a person can cut around 200 caps in an hour. I suggest that if you want to get many hundreds of caps that you simply have a cap-cutting jamboree - not so much biting the bullet as having a party. Seriously. I think you’ll have no problem getting volunteers and (COVID, etc. permitting), I’d be happy to come over myself.

    Best wishes,

    Jeremy

    Jeremy B.A. Green Ph.D.
    Vice-Dean International (Research)
    Professor of Developmental Biology
    Centre for Craniofacial & Regenerative Biology
    King's College London
    Guy's Tower, Floor 27
    London SE1 9RT
    UK

    jeremy.green at kcl.ac.uk
    Tel. +44 20 7188 1794
    https://www.kcl.ac.uk/people/jeremy-green

    On 29 Oct 2020, at 03:45, Leon Peshkin <peshkin at gmail.com> wrote:

    Dear Colleagues !

    I have a few general questions and tried posting to Slack "Xenopus Community" without success. People - Slack is a nice way to keep discussions going, please use it ! 

    Has someone worked out a way to make animal caps en masse ?  I did hear the gastromaster legend and even obtained one with a few tips … but it does not really work in my hands. Any other devices ?  Please do not tell me to just bite the bullet. I'd like to be able to cut many hundreds. 

       There are a couple of animal cap instructional videos with forceps but I know many people use other techniques. Would be nice to have more videos. 

    Does someone have a protocol and experience for blood perfusion in Xenopus ? 

       THANK YOU 

       - L. Peshkin 

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