strain expressing seam cell specific GFP marker?

Model Organism: Worms Methods & Reagents
1

Hi!

I am just starting to work with c. elegans and need a strain where GFP is expressed in the seam cells (scm::gfp seam-cell-specific marker) in order to be able to FACS them. Unfortunately such a strain is not available at the CGC (or at least I didn’t find one). Therefore I was wondering whether someone here in the community forum could help me out?

Thanks in advance!
Nadine

2

Hi,
You can search at http://gfpworm.org . If you search for ‘seam cell’ in the pattern search (selecting YES(or) in each life stage) then you get quite a few.
Here’s a link to the results http://tinyurl.com/4vv8xo

If you get the transgene name from the strain you can find further info about it in WormBase.
Not sure how you go about getting hold of these strains though.

Ant

3

Hi

The strain you are looking for is JR667 wIs51 and it is def available from the CGC.

Are you able to successfully get the seam cells out of the animals for FACS? I have often wondered if it would be possible to do this before! CAn you tell me how you disrupt the worms to get the intact cells out?

4

Hi,

The only way to get intact cells, is to culture cells from embryos. So that limits you to the cells that are born embryonically. If you disrupt the cuticle of larvae or adults en masse, then the cells just explode.

Good luck!

Clay

5

The Wormbook has a protocol for embryonic cell culture and FACS sorting, as well as references to published work describing the technique from multiple labs (David Miller, Strange, Jin, Chalfie, etc).

http://wormbook.org/chapters/www_intromethodscellbiology/intromethodscellbiology.html#d0e3828

Steve

6

Thanks for your help to you all! :slight_smile: Now I can get started…

But I do hope that there is a way to get intact cells out of larvae and adults, since this is what I would like to do. Why it should only work with embryos is not clear to me… Has anyone ever tried this before?

7

I’m sure if someone worked on isolating intact cells from postembryonic worms they would eventually succeed. As far as I know, however, this has not been vigorously pursued. The main problem is the tough outer cuticle. Whatever it takes to crack the cuticle ends up being more than the cells inside can take, and many of them burst open. If you wish to generate an expression profile of seam cells during postembryogenesis, you can use the mRNA-tagging method (See Roy et al 2002 and Von Stetina et al 2007).

Good luck!

Steve