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.
High Frequency Integrative Transformation 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. Injection Protocol My injection protocol is described in October 86 EMBO Journal -- here are some more suggestions: 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 a 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 still 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. X- gal 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 scopes. A Little More on HSP-Bgal expression 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 muscle 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.