From owner-celegans@net.bio.net Wed Oct 29 22:00:00 1997 Path: biosci!biosci!not-for-mail From: Thomas Burglin Newsgroups: bionet.celegans Subject: mating into vul. Date: 30 Oct 1997 06:15:21 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 191 Sender: daemon@net.bio.net Approved: wmorgan@acs.wooster.edu Distribution: world Message-ID: <63a4pp$kv0@net.bio.net> NNTP-Posting-Host: net.bio.net Hi, thanks for all the responses, which are summarized below. We are happy to report that we did manage to get an animal which crossed, so we don't need to resort to anything fancy. Nevertheless, it probably will occur in the future to someone out there that some reverse genetically engineered mutant has all kinds of other stuff in the background which might make backcrossing difficult. So here are various things to try. Best wishes Thomas ---------- Hey Thomas, I would suggest rescuing your mutant with the cloned gene. Once you have the strain rescued, you can cross away the array and maintain the vul mutation as a het. of course, injecting cloned gene will only work if your knockout causes this phenotype rather than some other randam mutation in background. Alternatively, the penetrance of some vuls is reduced following recovery through dauers or just recovery from starvation. If you see eggs on the plate under any condition, you can set of crosses with any animal that has not bagged yet. Even animals that do not lay eggs can be mated into if some vulva structural is present. This will not work with vuls like strong lin-39 alleles. Do the males mate? If so, just cross heat shock males into something like unc-58. Andy Papp claimed to poke holes into lin-29 animals and was able to mate into them. This might work if some vulva-like structure exists, but, I think, this approach will be futile for most things ( I did try once). Also, check the penetrance at different temps. Many things are weakly ts or cs. Finnally, why inject sperm if you can inject the cloned gene? Good luck. Scott Clark ------------- Hi Thomas, One question first - do you ever see eggs on a plate? If so, then some small number of animals is Egl+. So the first thing to try is putting 50 hermaphrodites down on a plate, for several plates, and keep your fingers crossed. Next thing to try is dauer. Many Vuls are rescued by going through dauer, so the way one mates into let-23 or lin-2,7 amd 10 is to starve a plate and then chunk and mate into the L4s that come out (again putting many animals down on the mating plate). If these don't work you may be out of luck in my experience. The only person I've ever heard of who got the 'poking a hole' method to work was Victor. I tried it myself with no luck. I do not know about injecting>sperm. Have you tried making heat shock males of the strain? If they mate (good chance they won't) you can try moving the mutation into another background that way (him-5 is always a good starting point). Also, since its a knockout have you tried rescuing it with the wild type gene? You could use rescued animals for crosses and then look for loss of the array (mark it with a GFP co-transformation marker). Its ugly but it works.> Can I ask what gene it is?? Hope this helps, David M. Eisenmann ----------- I don't know about state of the art, but I was able to mate a new lin-29 mutant (100% blip vulva, 0% mating and egg-laying) after perforating the vulva area by hand under the dissecting scope using a hand-held microinjection needle. Do several, and put them on a plate with lots of N2 males. I tried artificial insemination, too. That was worse, because you have to break off the needle to get the tip big enough to suck up sperm, and then it was too blunt and did too much damage to the p-vul recipient. Andy Papp ------------ Hello Thomas, When I was in Cynthia's lab Lisa Wrischnik successfully made LARGE holes in several lin-39 hermaphrodites and then smothered them with males. She did the operation on the injection scope and made quite large holes. I have had reasonable success transferring sperm from males to non-Vul hermaphrodites. Thus, I would try this first if you get males of your Vul (I am assuming they are me0). I tried collecting sperm from hermaphrodites but that was hard! Craig P. Hunter --------------- Thomas, as an alternative to poking holes in the worm and artificial insemination, you may, if the mutant is not too sickly, try heat shocking to increase the frequency of spontaneous males and then mate to N2 hermaphrodites or tra-2(q122) females. -Jim Waddle ------------- Hi thomas, Here's a hi-tech possibility. There are Muvs that are epistatic to a lot of vuls. You could try inactivating one of these by injecting oligos. May not work if your mutant transforms the Pn.ps out of the vulval equivalence group. If this is the case, getting males may not work either. Mike ---------------- And more info on injecting epistatic muvs: ----------- also, if you know what kind of vul, you could inject other genes to rescue, eg let-60, if not caused by your KO. there are not that many different kinds of real good vuls. if you are unlucky with trying the brute force approach to mating into and males do not mate (which is the case for many vuls) it might be worth looking at Pnp cells to see if you can quess the gene. eg lin-39 all 6 pnp cells fuse, no division, easy to score, cloned, easy rescue. determination vul, all 6 cells under 3* lineage, divide once, possible lin-2, 7, 10 (no L1 rod like lethality,) always less than 100% penetrant, try starvation recovery ,dauer, temp. lin-3, let-23, sem-5, let-341 let-60, vul and lethal let60 gf can suppress, thus can likely rescue with cloned let60 gene. lin45raf other downstream not usually strong vul mutations, but do have let. here is a long shot strategy, try lin-1 RNA inhibition. lin-1 downstream of all determination vuls, animals are muv, but may be able to mate into. at least it would be fun to try, lin25, heterchronic vuls, usually big protrusion at vulva. harder to deal with but many cloned, effort here gets to be a pain. my feeling is that if you see eggs on the plate, you are home free. i go through temp, starvation recovery, dauer recovery look for animals with some vulva like structure and less egl when rest are starting to bag. pick a bunch onto a plate, put down about 2x males, and it always has worked. but it would at fun to try other strategies, such lin1 rna. good luck scott ----------- Hi Thomas, The most downstream Muv that could be injected (dominant) would be something we call Vulvamaker in Stuart's lab, after Sevenmaker in Drosophila. It was made by Mark Lackner and he has spoken about it at meetings. It is an array that is a combination of 1) mpk-1 (MAP kinase) with an activating mutation in it (sufficient for activation in flies but strangely enough not in worms) and 2) MEK (MAP kinase kinase) driven from the HS promoter (I think). Apparently making more MEK allows the activated MAP kinase phenotype to show. This array, Ex or Is, makes worms very Muv, but in a temperature-dependent way - higher temp, more Muv. (This could be a problem if you have ts lethals in your background). I'm sure Stuart would give you the DNAs. Other than this the only other cloned activating mutations are in let-60 and let-23 (even more upstream). Good luck! Dave .