Biophysical and Cell Biological Approaches to the Cryopreservation of Embryos From Lines of Drosophila of Genetic Interest

通过精心控制俄勒冈R果蝇胚胎的发育阶段，我们成功地将它们置于低温下，并获得了相当高的存活率；约有65-70%的幼虫孵化，近40%的幼虫发育成可繁殖的成虫。拟议工作的目的是：(1)确定特别关键的因素及其容忍度，以便于果蝇社区使用该程序；(2)制定详细的程序规程，确定关键步骤及其公差；(3)对两个具有代表性的突变株系进行了适用性测试。关于目标(1)，我们发现限制存活的步骤是在玻璃化之前暴露于8.5 M乙二醇。这种接触造成的部分损害可能是由于加入浓缩乙二醇后的渗透性脱水造成的，也可能是由于玻璃化后去除乙二醇造成的渗透性肿胀。部分损害可能是所使用的高浓度乙二醇的化学毒性造成的。通过降低乙二醇的浓度，改变暴露温度，或使用渗透性更强的冷冻保护剂，如二甲亚砜，可以减少收缩的程度和持续时间，从而改善与收缩和毒性相关的损害，所有这些都将进行测试。要确定的一个关键点是在多大程度上可以操纵冷冻保护剂的这些方面而仍然实现玻璃化。为了减少渗透膨胀的潜在危害，我们将优化用于控制渗透膨胀的蔗糖稀释程序。我们还建议确定玻璃化过程中某些关键步骤本身的公差。这些包括(a)沸腾的液氮可以代替氮泥吗？如果是这样，它将简化程序；(b)现在使用的15小时胚胎是否与以前使用的12小时胚胎一样，对-100摄氏度以上的短暂暴露和比超快速变暖速度慢的变暖速度敏感？这些方面的知识对程序的使用者是很重要的。由于黑腹果蝇在实验中是如此强大的生物体，突变体和/或转基因种群的数量已成为一个压倒性的问题。迄今为止，所有库存必须每两周转移一次。冷冻胚胎的方法非常简单，可以在生物实验室中使用，并且可以将受精率恢复到40%的水平，这种方法的发展将在相当短的时间内节省数百万美元和工时。它还应该有助于确保重要的种群不会随着时间的推移而发生遗传漂变。

By carefully controlling the developmental stage of Oregon R Drosophila embryos, we have succeeded in subjecting them to cryogenic temperatures with reasonably high survivals; Some 65-70% hatch and nearly 40% of the resulting larvae develop into fertile adults. The purpose of the proposed work is (1) to identify the especially critical factors and their tolerances so as to facilitate the use of the procedure by the Drosophila community; (2) prepare a detailed protocol of the procedure which identifies the critical steps and their tolerances; and (3) test the applicability of the procedure to two representative mutant lines. With respect to Aim (1) we are finding that the step that limits survival is the exposure to 8.5 M ethylene glycol that precedes vitrification. Part of the damage from that exposure could be a result of the osmotic dehydration accompanying the addition of concentrated ethylene glycol or it could be osmotic swelling associated with the removal of ethylene glycol after vitrification. Part of the damage could be the result of chemical toxicity of the high concentrations of glycol used. The damage associated with shrinkage and toxicity could be ameliorated by reducing the extent and duration of shrinkage by reducing the concentration of glycol, by changing the exposure temperature, or by the use of a more permeable cryoprotectant like perhaps dimethyl sulfoxide, all of which will be tested. A critical point to be determined is to what extent one can manipulate these aspects of the cryoprotectant and still achieve vitrification. To reduce potential damage from osmotic swelling, we shall optimize the sucrose dilution procedure used to control that swelling. We also propose to determine the tolerances to certain critical steps during vitrification per se. These include (a) can boiling liquid nitrogen be used in place of nitrogen slush? If so, it will simplify the procedure; (b) are the 15-hr embryos now being used as sensitive to brief exposures above -100 C and to warming rates slower than ultra rapid as the 12-hr embryos used previously? Knowledge on these points will be important to users of the procedure. %%% Because Drosophila melanogaster is such an experimentally powerful organism, the numbers of mutant and/or genetically modified stocks has become an overwhelming problem. To date, all stocks must be transferred about every two weeks. The development of a method to freeze embryos which is simple enough to be used in biology laboratories and yet allows a recovery of fertile at a 40% level will save millions of dollars and man-hours of work in a reasonably short time. It should also help to ensure that important stocks do not undergo genetic drift over time.