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州R的发育阶段， 果蝇胚胎，我们已经成功地将它们置于 低温，具有相当高的存活率;约65-70% 孵化，近40%的幼虫发育成可育的 成年人了 建议工作的目的是（1）确定 特别是关键因素及其公差， 促进果蝇群体使用该程序; (2)准备一份详细的程序协议， 关键步骤及其公差;（3）测试 该程序对两个代表性突变株系的适用性。 关于目标（1），我们发现限制的步骤 存活率是暴露于8.5 M乙二醇， 玻璃化 暴露的部分伤害可能是 渗透脱水的结果伴随着添加 浓缩的乙二醇，或者它可以是渗透溶胀 与玻璃化后乙二醇的去除有关。 部分损害可能是化学毒性的结果， 使用更高浓度的乙二醇。 与之相关的损害 收缩和毒性可以通过降低 和收缩的持续时间， 通过改变暴露温度，或者通过使用更 渗透性冷冻保护剂，比如二甲基亚砜， 这将被测试。 要确定的一个关键点是， 在一定程度上可以操纵冷冻保护剂的这些方面， 仍然实现玻璃化。 为了减少潜在的损害， 渗透膨胀，我们将优化蔗糖稀释程序 用来控制肿胀 我们还建议确定 玻璃化过程中某些关键步骤的公差。 这些问题包括：（a）是否可以使用沸腾的液氮来代替 氮气泥浆 如果是这样，它将简化程序;（B）是 15小时胚胎现在被用作对上述短暂暴露敏感的胚胎 -100 C和升温速度慢于超快速的12小时 以前使用过的胚胎？ 这些知识将 这对程序的用户来说很重要。 %%% 因为黑腹果蝇在实验上是如此强大 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，所有库存都必须 大约每两周转一次 开发一种方法， 冷冻胚胎，这很简单，可以用于生物学 实验室，但允许在40%的水平恢复肥沃 将节省数百万美元和工时的工作在一个合理的 短时间 它还应有助于确保重要的种群 不会随着时间的推移发生基因漂移。

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.