Stanley P. Leibo

A two-step method for permeabilization of Drosophila eggs☆

As a first step in developing a procedure for the cryopreservation of Drosophila melanogaster embryos, we have established a method for permeabilization of the eggcase and have initiated studies of the hydraulic conductivity of permeabilized embryos and the permeation of selected cryoprotective agents. The eggcase of D. melanogaster embryos has a wax layer that precludes any flux of water. A two-step procedure employing organic solvents was developed to effect removal of the wax layer with minimal deleterious effects on the embryos. Dechorionated embryos (Oregon-R strain P2, 12 to 13 hr old) were rinsed sequentially in isopropanol and hexane. After removal of solvent, embryos were held in a modified cell culture medium for further manipulation. This procedure routinely yielded 80 to 95% of the eggs permeabilized (as determined by osmotic contraction in 1 M sucrose) and 75 to 90% survival (incidence of hatching). Hydraulic conductivity of permeabilized embryos and permeation of cryoprotectants were determined using a microdiffusion chamber and computerized video microscopy. Regression analysis of the volumetric data from individual embryos yielded the Boyle-vant Hoff function FVeq = 0.124 (osm-1) + 0.541 with the standard deviations of slope and intercept (Vb) being 0.010 and 0.040, respectively. Permeabilized embryos exhibited ideal osmotic behavior over the range of 0.265 to 2.00 osm. The mean hydraulic conductivity coefficient (Lp) was 0.722 +/- 0.366 micron/(min.atm) at 20 degrees C, based on observations of contraction following a step change in concentration of Ringers solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryobiology of Drosophila Melanogaster Embryos

The common fruit fly Drosophila melanogaster is the subject of investigation in many diverse areas of biology. It has been studied intensively by geneticists, developmental and molecular biologists, neurobiologists, population and evolutionary biologists, entomologists, and chronobiologists. Currently, interest in D. melanogaster is most intense among molecular biologists, but studies of D. melanogaster have a long and distinguished history, dating back to Thomas Hunt Morgan in the first decade of this century. As a result of both past and present activity, there is an enormous number of D. melanogaster genetic stocks. In 1985 it was estimated that the number of different stocks was in excess of 30,000 and was rapidly increasing because of the increased number of investigators studying Drosophila, the increased number of large scale mutant screens, and the generation of new stocks by DNA transformation. Since then, the number of mutant stocks is even greater, especially since so many germ line transformants have been obtained; for example, in Drosophila Information Service (June 1988), some 1350 entries were recorded in the “clone list.” Many of these clones have been reinserted in several different places in the germ line via P-element mediated transformation. We estimate that over 50,000 different genetic lines of D. melanogaster are now maintained in national and international stock centers and in the laboratories of individual investigators.