Use of the Laser Microbeam for Preserving Frozen Drosophila Embryos : Cell Biology

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The possibility of using the laser microbeam to preserve early embryos of Drosophila melanogaster at -196℃ was examined. To date, the most difficult problem encountered in freezing Drosophila embryos has involved the method by which a protective agent is introduced into the eggs through the vitelline membrane. In the present study, a UV laser microbeam was applied to inflict breaks or cuts on the micropile of the eggs in order to allow protective glycerol to enter. Specifically, the laser was directed against the micropile of dechorinated eggs, after which the eggs were incubated at 25℃ in Medium K-17 supplemented with fetal bovine serum and glycerol. Following a 1-24hr incubation, the eggs were frozen rapidly at -196℃ in liquid nitrogen. After at least another 24hr, the eggs were thawed and incubated in a physiological salt solution at 25℃. A few of the eggs hatched, and the larvae grew to adult flies. Glycerol incorportation was comfirmed by staining the eggs with neutral red. The survival rate of frozen, irradiated eggs increased upon incubation with glycerol for at least 16hr. The stage and site of eggs to be laser irradiated, the conditions of incubation, and the rates of freezing and thawing are now under investigation so as to yield a larger number of viable eggs.
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