Worm Breeder's Gazette 9(3): 76

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Injection Miscellany

A. Fire

High Frequency Integrative 
I have been recently been obtaining higher frequencies of 
integrative transformation than those I described in previous gazette 
articles. The highest frequencies of transformation have come from 
experiments where I have injected oocyte nuclei in the proximal arm of 
very young adults (i.e adults which have fertilized only 1 to 3 
oocytes). The current rate (with sup-7 injected into tra-3[am]) is one 
transformant in 5-10 injected worms [all accesible oocytes in each 
worm are injected]. Work per injected line has decreased 
proportionally to about 4 hr of 'real work' per transformed line.
My injection protocol is described in October 86 EMBO Journal -- 
here are some more 
Rather than gently pushing the needle into the worm (which seems to 
greatly decrease viability) it is much healthier to drive the needle 
in with 
rather VIOLENT 'whack' at the micromanipulator or microscope. The 
worms dont seem to mind this at all and can stand dozens of such 
injections and 
The visibility of structures in the microscope provides a good assay 
for whether the animals to be injected have drled down too much on 
their agarose surface. If the proximal oocyte nuclei become invisible 
under the plan 40 nomarski, your worm is too dry. If this happens in 
less than ten minutes after the worms are mounted, then you should try 
thinner agarose pads (it will happen after a long time even with pads 
of the correct thickness, when starting out it is best to put only as 
many worms on a single pad as can be injected in about 10 minutes). If 
the worms are still moving around after 3 or 4 minutes, your pads are 
too thin, or the intermediate plate between transferring worms from 
their home to the pad was not sufficiently dry.
Some batches of Voltalef 3S oil don't let worms stick permanently (
they stick and then come off). All other batches of 3S oil seem 
equivalent. (Several good batches of oil have been obtained at the MRC 
and good batches have also been obtained in Bloomington [J. Cane] and 
the Bronx [K. Nelson]). Heavy parafin oil (BDH product 29437 or 
equivalent) can also substitute for Voltalef 35 oil in the injection 
procedure without any obvious difference in the handling of the worms 
or in subsequent fertility, so there doesent seem to be anything 
magical about Voltalef oil.
If you think you are having trouble obtaining oil that allows your 
worms to stick down to dry agarose pads, send me a small sample of 
your oil (lOOul) to the Carnegie and I will test it. If there is a 
problem I can send a small sample of a good batch.
DNA concentration dependences(based on experiments done in 
collaboration with J. Cane and T. Blumenthal, and with R. Waterston)
Integrated transformants have now been obtained at a variety of DNA 
concentrations from 60 ug/ml to 2 mg/ml. There doesn't seem to be huge 
dependance on the DNA concentration in this range but more data would 
be very useful on this point. Cosmids get difficult to inject at the 
high concentrations.
I have switched to X-gal staining of worms carrying B-gal fusions. (
This seems more sensitive and avoids the nasty azo-dye coupling 
reagents used in the napthyl protocol.) A few crystals of X-gal are 
stuck in the bottom of a 1.5 ml microfuge tube and dissolved in lOul 
of dimethylformamide. One ml of 0.2M NaPhosphate pH 7.5 and 100 ul of 
oxidation buffer [50 mM KFerricyanide+50mM KFerrocyanide] are added, 
and the tube is vigorously mixed and spun in a microcentrifuge to 
pellet excess Xgal. The supernatant is added to 8ul of 1%SDS and added 
to pipetted onto worms that have been vacuum and acetone fixed as I 
described in the last newsletter (the acetone step is actually not 
necessary). In order to detect very weak signals, it is important to 
incubate the slides at 37 C. The Leitz dissecting microscopes give 
poor color resolution for seeing light blue stains; resolution is much 
better under the bright-field illumination of the Zeiss high power 
A Little More on HSP-Bgal 
CB4027 is a strain containing multiple integrated copies of a fusion 
between a D. italicter heat shock promoter region and E. 
coli B-gal (see last news letter).
Histochemical staining of CB4027 animals heat shocked for different 
amounts of time reveals that the response turns on first in early 
embryos (after only 1 hr at 34 C) and only later in the pharynx (after 
>2 hr.) Also heat-shocked CB4027 late embryos have been stained with a 
polyclonal mouse antibody to B-gal (the antibody gives only background 
staining on unshocked CB4027 or wild type embryos). The staining in 
these embryos is most prominent in the gland cells in the pharynx, and 
in two cells in the tail, which only turn on the B-gal when the embryo 
is in the 2-2 1/2 fold stage.
Expression of a MyoII-Bgal fusion in pharyngeal 
Myo-2 is one of the two myosin isoforms that had been shown to be 
specific to the pharyngeal muscles. A fusion between the putative 
myoII promoter (from Ichi Murayama's sequence) and the B-gal coding 
region was constructed. This was introduced by cotransformation with 
sup-7. One of the resulting lines expresses the E. coli B-gal. When 
these animals are fixed and stained with X-gal as described above, 
staining appears only in the pharyngeal muscle. Both the suppression 
and B-gal activities breed true, a homozygote line has been 
established, and the pharyngeal B-gal activity and amber suppression 
activities cosegregate in outcrosses of this line.
Although both B-gal constructs so far tested lead to pharyngeal 
staining, the pattern of B-gal expression in myoII-Bgal line is 
different from the pattern with the HSP-Bgal fusion construct: 
pharyngeal staining of animals transformed with the HSP-Bgal construct 
does not appear to be localized within muscle cells (I'm still not 
sure exactly what the localization in larvae and adults carrying HSP-
Bgal is, but see above). Also, expression of the HSP-Bgal construct 
absolutely requires heat shock and also occurs in early embryos (early 
embryos are not stained in animals carrying the myoII-Bgal fusion).
Obviously it would be nice to look at B-gal fusions using many 
promoters with a variety of different localizations. This could 
provide the best demonstration that patterns of tissue specific Bgal 
staining accurately reflect differences in promoter function rather 
than some odd properties of the B-gal segment.