Inability to establish a cross between roller hermaphrodite CL2006 and males

Model Organism: Worms Methods & Reagents
1

Hello,

I am new to this research area and I am presently working on some of the experiments where I am required to establish genetic crosses. I am trying to establish a cross between CL2006 (Roller) Hermaphrodite and QC 47 males. I tried heat shock method as well as alchohol exposure method. During the last attempt I kept 10 males along with twe CL2006 hermaphrodite. But all the rollers lacked QC 47 expression. I also observed the worms under microscope and found that males were not reluctant in approaching cl2006 hermaphrodite. Notoriousy in one of the plates i poked the males to approach CL2006hermaphrodites and even dragged them near to CL2006 . So far I have been unsuccessful in establishing the cross. Please help with your valuable suggestion.

Thanks

2

Hi,
In my honest opinion, this cross has really low chance of working out becz of the crazy and kind of uncrossable behavior of the roler folks. I have tried this myself, 3 times and it just didint work out.
So, you should look for alternatives around the available strain or using recombennering to make your own contruct with gene of interest in a normal worm and then using it to cross.

Sorry about that…but thats my experience and opinion. ::slight_smile:

3

If I understand correctly, both your males and your hermaphrodites are Rollers. If you males were not Rollers you wouldn’t have any problem. So there are a couple ways to try to get Roller males to mate:

  1. Put an absurd amount of males on plate in relation to hermaphrodites. 40 males to 4 hermaphrodites, for example. You may get lucky.
  2. Feed Roller males (and your hermaphrodites too, although not necessary) on rol-6 RNAi. As long as your RNAi isn’t superstrong you should get some males/hermaphrodites that do not Rol. If your RNAi is too strong you’ll go the other way and have too little WT rol-6 and they’ll Rol. It isn’t as finicky as it sounds, and it’s worked for us.

Good luck!

4

Further to Steve Von Stetina’s comment, one good way to make sure you’ve got lots of males carrying etIs2 from QC47 would be to set up a mating stock of N2, and cross those males into QC47 hermaphrodites to get lots of etIs2/+ males to cross into CL2006, with or without growing those males on rol-6(RNAi) bacteria.

You could also use him-5 males at the first step, but doing it that way will likely leave him-5 floating around in the double-transgene strain you construct, which you may not want.

5

Hi every body

First of all thanks for response and helping me. The QC47 male are not rollers only cl2006 hermaphrodies are rollers. I am actually concerned to know , what is the possibility of such crosses to occur. Is it the case that normal males don’t tend to approach roller hermaphrodites. Because when I dragged one of the male to Cl2006 hermaphrodite it immediately moved away. one more thing whenever I used to observe the plate under microscope I never found males near to hermaphrodites. Please give your valuable suggestion

6

I am a little confused why your QC47 males are not Rollers. According to the CGC the genotype of QC47 males is:

Genotype: etIs2 III.
Description: pRF4 + pRIC-19::GFP. Expressed the RIC-19::GFP fusion protein under the ric-19 promoter in all neurons weakly, except for strong expression in the pharyngeal M2 neurons

pRF4 (dominant rol-6) was used to make the line, so in theory they should Rol.

So I will ask you: are you sure what you are calling QC47 is the correct genotype? Do they have the GFP expression you expect? Because if your starting strain does not express, your cross-progeny never will!

I have rarely had problems generating cross-progeny from a non-Rol male and a Rol hermaphrodite. Do you have any males produced as cross-progeny from the cross? That’s a sure-fire way to know if the cross has been successful.

Perhaps the problem is your hermaphrodite strain, however. According to the CGC this is the genotype of your hermaphrodites:
dvIs2[pCL12(unc-54/human Abeta peptide 1-42 minigene) + pRF4]. Adult onset paralysis and egg-laying deficiency when raised at 20C.

Are you doing your cross at 20C? Does the paralysis/Egl phenotype not present at 15C? If so, perhaps you should try the cross at 15C.

Sorry for rambling - hope you can get this to work!

7

thanks again

My QC 47 moves normally but it does show neuronal gfp expression.
Perhaps you have pointed the correct loop hole . I have been trying to cross it at 22 degrees and I believe I should try to cross it at 15 degrees may be it will solve the problem

Thanks

8

Quick suggestion…

Increase your number of males. like atleast 15 to 1 hermaphrodite.

Have your E. coli lawn small… so that the worms tend to stay only on that small portion of the plate, not throughout the plate.

Keep your mating plate @ 15C, not a higher temperature.

Hope this works.