mating into vul.
burglin at ubaclu.unibas.ch
Thu Oct 30 09:15:21 EST 1997
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.
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
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
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?
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
(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.
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.
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.
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
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
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.
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).
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