SUMMARY: transgenics in other nematodes

Leon Avery leon at eatworms.swmed.edu
Thu Jul 6 12:58:56 EST 2000


My query about getting transgenes into nematodes other than C elegans 
elicited both positive and negative responses.  First, lots of people 
said C briggsae works -- I won't include any of the individual 
messages.  The other clear positives are Heterodera glycines (David 
Bird), Heterorhabditis bacteriophora (Randy Gaugler), and Panagrellus 
redivivus (Barbara Meyer).  rol-6 is reported to work in C briggsae 
(which doesn't surprise me) and P redivivus (which does).  The 
negatives are Parastrongyloides trichosuri (Chuck Shoemaker; I gather 
that it may simply be mechanically difficult to do the injections in 
this species) and Oscheius sp. CEW1 (Marie-Anne Felix).  Ralf Sommer 
also sent a brief, amazingly cryptic note saying that "Transgenics is 
a problem in all non-Caenorhabditis nematodes."  He didn't say what 
problems he'd had or which species he'd tried, but implied 
Pristionchus was one of them.

Several people stated that transgenes would not be transmitted unless 
chromosomes are holocentric, and that we don't know whether 
chromosomes are holocentric in other nematodes.  This sounds 
plausible, although I suspect that the proposed relationship between 
holocentricity and transgene transmission is based on assumptions 
that have not actually been experimentally tested.

--
Leon Avery                                       (214) 648-4931 (voice)
Department of Molecular Biology                           -1488 (fax)
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd                            leon at eatworms.swmed.edu
Dallas, TX  75390-9148                  http://eatworms.swmed.edu/~leon/

POSITIVES:

Hi Leon, Yes, we did make panagrellus transgenics by the usual 
injection route. We had no problems getting rollers with the dominant 
rol injection marker.
Barbara
________________________________
Barbara J. Meyer
Investigator, Howard Hughes Medical Institute
Professor of Genetics and Development
Department of Molecular and Cell Biology
401 Barker Hall
University of California-Berkeley
Berkeley, CA 94720-3204
phone: (510) 643-5585
fax: (510) 643-5584
e-mail: bjmeyer at uclink4.berkeley.edu

Leon,
we have made transgenic soybean cyst nematodes (Heterodera glycines). 
Because of the biology (the females aren't transparent, and are 
inside the host plant for much of the time), we injected males (the 
testis). Injected males were then crossed, and the progeny scored. We 
found that the C. elegans myo-3 promoter driving gfp (from And's kit) 
worked well. We obtained transgenic progeny from each injection 
(well, we did it 3 times), and about 70% of the brood expressed the 
reporter in each case. We presumed that extrachromosomal arrays 
formed, but we really haven't followed up on this (each experiment 
takes several months, and requires maintaining the worms inside 
plants). This is not published, but you are most welcome to use C H 
Opperman and D M Bird as a pers comm in your proposal if it would 
help.
dave
David McK. Bird
Associate Professor of Plant Pathology
Associate Director, Center for the Biology of Nematode Parasitism
Plant Nematode Genetics Group
Box 7616, NC State University
Raleigh, NC 27695-7616

Hi Leon,
I had heard through the grapevine that some of the nematologists at NC
State (Opperman, maybe...) had gotten in working in the plant parasitic
nematode H. Schactii. You might want to check with them if you don't hear
from them directly, that is.
Cheers,
rob
Robert Rutherford, Ph.D.
Postdoctoral Fellow
Program in Developmental Biology
Department of Anatomy-UCSF
Phone (415) 476-3557
Fax (415) 476-3493

Dear Leon,
I haven't done this, but Randy Gaugler's lab did it with the
entomopathogenic nematode Heterorhabditis bacteriophora. They used rol-6dm
and a hsp-16/lacZ reporter. Reference is: J. Invertebrate Pathology
66:293-296 (1995). Hope this helps.
Creg Darby

NEGATIVES:

Transgenics is a problem in all non-Caenorhabditis nematodes. In
Pristionchus we are just learning that the difference in gonad morphology -
as seen by TEM - might play a crucial role. It is unclear however, if that
is the only problem.
Ralf
___________________________________________________________________________

Ralf J. Sommer, Dr., Priv.-Doz.
Max-Planck Institute for Developmental Biology
Dept. for Evolutionary Biology
Spemannstrasse 35
D-72076 Tuebingen
Germany
Phone: ++49 7071 601371
Fax: ++49 7071 601498
Email:ralf.sommer at tuebingen.mpg.de

Leon,
In my group here in NZ, we have been attempting to make transgenics by gonad
injections in Parastrongyloides trichosuri (a worm parasite in possums that
has a free-living cycle in feces that we can reproduce in culture). Using
standard techniques we can perform successfully in C. elegans, we have so
far been unsuccessful in generating transgenic Pt. There are technical
problems relating to the "fragility" of the worm and the lack of
hermaphrodites in this worm that could explain our failure to date although
we remain optimistic we will overcome these problems. We are also thinking
of trying the "microprobes" used successfully with Heterorhabditis
bacteriophora (BioTechniques 1995, 19:766). I will be interested in hearing
about what you learn in response to your email if possible, and how others
have fared with microprobes if you know.
Chuck Shoemaker


Dear Leon,
Unfortunately, it does not appear to be so easy to do transgenics in other
species (except briggsae), even in the family to which C. elegans belongs
(for example in Oscheius sp. CEW1 which we study here in the lab). There
may be many reasons why it does not work (accessibility of nuclei,
formation and segregation of an extrachromosome, etc.). We haven't found
out a solution yet. Maybe direct integration or transposition.
There are reports of transformation of the insect parasite Heterorhabditis.
for example Journal of Experimental Zoology 281: 164-170 1998
Best wishes,
Marie-Ahne


Marie-Anne Felix
Institut Jacques Monod, Tour 43, 2 pl. Jussieu,
75251 Paris Cedex 05, France
Tel: +33-1-44-27-40-88; Fax: +33-1-44-27-52-65
(5e etage, couloir 42-43)

--
Leon Avery                                       (214) 648-4931 (voice)
Department of Molecular Biology                           -1488 (fax)
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd                            leon at eatworms.swmed.edu
Dallas, TX  75390-9148                  http://eatworms.swmed.edu/~leon/


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