The ultimate goal of this project is to develop uncomplicated low- cost protocol for the cryopreservation of Drosophila melanogaster embryos. To achieve this goal, three strategies of cryopreservation will be investigated: 1) """"""""conventional"""""""" cryopreservation produces involving the incorporation of cryoprotectants (CPA's) such as DMSO, glycerol, ethylene glycol and a two-step cooling protocol (controlled cooling to effect freeze-induced dehydration followed by quenching in liquid nitrogen, LN2); 2) """"""""vitrification"""""""" procedures which involve the use of highly concentrated mixtures of cryoprotectants (DMSO, acetamide, propylene glycol and polyethylene glycol) which preclude ice crystallization in the system during cooling in LN2; and 3) """"""""supercooling"""""""" procedures which involve storage at low temperatures (ca -20 degree C), which are low enough to inhibit metabolic activity but above the embryo nucleation temperature. For the conventional cryopreservation approach, studies will be directed to characterizing the chilling sensitivity of embryos as a function of developmental stage; development of protocol for the permeabilization of the eggshell for flux of water and CPA's; determination of the hydraulic conductivity and solute permeability coefficients; computer-enhanced video cryomicroscopic studies of the cryobehavior of embryos during a freeze/thaw cycle to determine the volumetric behavior and nucleation temperature as influenced by the permeabilization protocol, the composition of the suspending medium, and the cooling/warming protocol; modelling of the thermodynamics of mass transfer during freeze-induced dehydration; and characterization of the phase behavior of the embryo cytosol by DSC (differential scanning calorimetry) and high resolution cryomicroscopy. For the """"""""vitrification"""""""" approach, studies will be directed to the optimization and utility of vitrification solutions and procedures for Drosophila embryos. For the """"""""supercooling"""""""" approach, studies will be directed to the characterization of an optimum suspending medium and will contrast the effectiveness of """"""""fluoro-inerts"""""""" to mineral oils. Additionally the temporal stability of the metastable state and assessment of metabolic deterioriation at -20 degree C will be investigated. Studies of the long-term viability and genetic stability will follow formulation of an appropriate cryopreservation protocol. A multidisciplinary team has been assembled for this work and includes two Drosophila biologists/geneticists, three cryobiologists with considerable practical experience in the cryopreservation of mammalian embryos and the cellular and molecular aspects of freezing injury, an engineer for the modelling studies, three research associates experienced in the areas of computerized video cryomicroscopy, membrane biochemistry, lipid analysis, DSC, and electron microscopy.
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