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

通过仔细控制俄勒冈R果蝇胚胎的发育阶段，我们成功地将它们置于低温下，获得了相当高的存活率；一些65%-70%的孵化和近40%的幼虫发育成了可生育的成虫。拟议工作的目的是(1)确定特别关键的因素及其耐受性，以便于果蝇群体使用该程序；(2)制定该程序的详细方案，确定关键步骤及其耐受性；以及(3)测试该程序对两个具有代表性的突变系的适用性。关于AIM(1)，我们发现限制生存的步骤是在玻璃化冷冻之前暴露于8.5M乙二醇组。暴露造成的部分损害可能是伴随着添加浓缩乙二醇而导致的渗透脱水，也可能是玻璃化冷冻后乙二醇被去除而导致的渗透性肿胀。部分损害可能是高浓度乙二醇所造成的化学毒性的结果。与收缩和毒性相关的损害可以通过减少乙二醇的浓度、改变暴露温度或使用渗透性更强的冷冻保护剂(如二甲基亚砜)来减少收缩的程度和持续时间，所有这些都将进行测试。需要确定的一个临界点是，一个人可以在多大程度上操纵冷冻保护剂的这些方面，并仍然实现玻璃化。为了减少渗透肿胀的潜在损害，我们将优化用于控制渗透肿胀的蔗糖稀释程序。我们还建议确定玻璃化过程中某些关键步骤本身的公差。这些问题包括：(A)可以用沸腾的液氮代替氮泥吗？如果是这样的话，它将简化程序；(B)现在使用的15小时胚胎对零下100摄氏度以上的短暂暴露和升温速度是否像以前使用的12小时胚胎那样对超快速度敏感？对这些要点的了解对该程序的使用者将是重要的。%由于黑腹果蝇在实验上是如此强大的有机体，突变和/或转基因种群的数量已经成为一个压倒性的问题。到目前为止，所有库存大约每两周就必须转移一次。开发出一种冷冻胚胎的方法，这种方法足够简单，可以在生物实验室使用，但可以在40%的水平上恢复受精率，这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股票不会随着时间的推移而发生基因漂移。

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.