Starting with C. elegans - special equipment needed? (Dec/06/2006 )

Next year we like to do some experiments with C. elegans;

we are fully equipped for normal cell culture and microscopy but I have no idea how to culture C. elegans. Is it difficult for beginners? Is there something special needed - beside the worms of course? How to feed?

---

Hi there!

I worked in a c. elegans lab once. they had special incubators (20°C), you keep the worms on agar (think it was NGM I Medium, quite simple but with a lot of additional solutions, one of the is Nystatin against fungus, very important). Feeding: E. coli OP50, you just drip liquid culture on agar plate, incubate 24 h 37°C and then worms on it. For proper experiments you need to synchronise worms by doing egg preparation (protocol in lab animal forum), for "culture propagation" all you nedd to do is cut a little bit of agar out from old plate, put it on new plate.

what exactly do you want to do with C. elegans?

More info on C elegans: http://elegans.swmed.edu/

Stardust

---

Hay stardust, thanks a lot

we have some fluorescent protein tagged proteins and like to express and oberve them in C. elegans

I think normal confocal microscopy will do or is two-photon microscopy necessary (we are also equipped with this)?

I read that transfection of C elegans is easy as you give your plasmids to their feed. Is that right?

we like to watch distribution and redistribution of our proteins. what can also be analyzed for C elegans? I suppose that behavior (motility) must be an option; are there C elegans specific physiological tests or tests of behavior? or any other standard tests in this field?

---

Hi kosmodrom,

We were interested in the behaviour of different bacteria mutants in the gut of C elegans. So we had bacteria with plasmids expressing EGFP or DsRed in the bacteria and used Laser scanning microscopy to visualize in the gut. I don't know if you can just "transfect" them by feeding, for siRNA (long dsRNA) the just soak the worms in the liquid containing dsRNA. With plasmids i don't know because we used plasmids active in the bacteria we feed them and i don't know if the DNA can "leave" the bacteria and get taken up in the worm. Analysis in my ex institute involved killing assays (find out which bacterial mutants are more "toxic") and LSM of bacterial distribution in the gut. Motility is always an option. Some test are performed on different media like NGM II and PGS. After working with the worms a bit you can see differences by eye, like some get defects in laying eggs or so, some move different. I have no idea what your protein is or what you hope to observe so i don't know which tests are suitable

Stardust

---

thanks very much again stardust for competent reply;

we like to modify signal transduction pathways of cells by transfecting kinases, Y-kinase receptors and phosphatases; as I see it must be difficult to transfect tissues specifically; what about transfecting the eggs? then, some adults may express our transfected proteins in every tissue, am I right?

---

eggs are not easy to transfect i think, they are surrounded by a "shell" e.g. if you want to isolate eggs you destroy the worms with bleach but nothing happens to the eggs


found this in a paper:

2.7. Transformation of C. elegans

Parasite genes (Grant, 1992; Roos and Grant, 1993) and
their promoters (Kwa et al., 1995; Qin et al., 1998; Britton
et al., 1999; Krause et al., 2001; Redmond et al., 2001) can be
analysed in transgenic C. elegans. A reporter construct
consisting of 1.3 kb of sequence upstream of the predicted
initiation codon of the Pt-hsp-1 gene was cloned into a
modified pPD95 vector (Fire et al., 1990) to drive
the expression of a b-galactosidase/green fluorescent protein
(b-gal/GFP) fusion reporter protein. This plasmid (designated
pPtHsp70-prm) was microinjected into the gonad of
young adult N2 C. elegans hermaphrodites with the dominant
rol-6(su1006sd) transformation reporter (Mello et al., 1991)
and transgenic rolling lines established by cloning of rolling F1
progeny of the injected worms. Transgene expression was
detected by staining for b-gal activity.

---

thanks so much stardust, I will try our micromanipulator for direct injection of DNA...

---

I work with C.elegans now, and have been working with them for years. If you have questions message me. In your message include your e-mail so we can write directly about it. C.elegans is a very simple organism. Growing them is easy. Just leave them at 20C. If the plates are old and crowded they can dauer, which is an arrested development state, simply put on new plates and they will continue to grow. They are hardy and hard to kill. Just practise moving them.

---

thank you very much nmstew, when I start there will be likely some questions, it is good to feel free someone to ask who is competent

---