CRYOBIOLOGICAL PRESERVATION OF ANOPHELES EMBRYOS

疟疾每年感染超过2亿人，并导致近 200万人，其中大部分是儿童。传播学的许多方面 疟原虫是受按蚊遗传影响的寄生虫。 因此，昆虫的分子遗传学是一门 密集的研究。一个具体的研究领域是培育突变系。 携带有阻止疾病发展或传播的基因的按蚊 寄生虫，会在本土蚊子中传播的基因 当被引入该人口时，该人口。不管是为了这个 特定目的或更广泛地说，分子遗传学方法通常 需要创建和分析数百条候选线以 获得一个具有所需遗传特征的人。发展的步伐 这种研究受到严重阻碍，因为即使是大型实验室也无法 同时保持几个以上的繁育系。那个障碍 如果冷冻保存成为可能，它们将在很大程度上消失。这项建议 关心的是实现这一目标。成功的可能性一直是 最近成功地冷冻保存了果蝇胚胎，这一点得到了加强。然而， 尽管按蚊和果蝇的胚胎在低温生物学上表现出 相关相似之处，我们最近的研究表明 重要的区别。为了在低温下存活，细胞必须 对水和低温保护溶质都具有渗透性。无论是果蝇还是果蝇 也没有按蚊胚胎满足这一要求。果蝇和按蚊可以 两者都可以通过接触酒精和烷烃而变得可渗透。虽然 对于前者是无害的，但不幸的是，这种治疗对 后者是因为，自相矛盾的是，它们的透水性太高。我们 然而，现在已经实现了渗透性幼年按蚊的存活 通过用风干代替酒精来培养胚胎。第二个严重问题 果蝇和按蚊都表现出高度的敏感度 甚至在没有冰冻的情况下也会变冷。对冷害高度敏感 通过要求更高的玻璃化来迫使一个人进行冷冻保存 接近。较老的按蚊胚胎对冷的敏感度要低得多，但它们 对年轻人有效的渗透过程不起作用 胚胎。我们提出了一些方法来规避对寒冷敏感的 更年轻的胚胎，我们提出了一些顺序的物理和 使较老的胚胎渗透的化学方法。体能 方法包括用阿尔法粒子轰击和电穿孔。 化学方法包括用超临界二氧化碳萃取。

Malaria infects over 200,000,000 people annually and kills nearly 2,000,000, mostly children. Many aspects of the transmission of the Plasmodium parasite are under genetic influence of the Anopheles mosquito. For this reason, the molecular genetics of the insect is a subject of intensive research. One specific research area is to develop mutant lines of Anopheles with genes that block the development or transmission of the parasite, genes that would spread throughout the indigenous mosquito population when introduced into that population. Whether for this specific purpose or more generally, the molecular genetic approach usually requires the creation and analysis of hundreds of candidate lines to obtain one possessing the required genetic characteristics. The pace of such research is severely impeded by the inability of even large labs to maintain more than a few breeding lines concurrently. That impediment would largely disappear if cryopreservation were possible. This proposal is concerned with achieving that goal. The likelihood of success has been enhanced by recent success in cryopreserving Drosophila embryos. However, although Anopheles and Drosophila embryos exhibit cryobiologically relevant similarities, our recent research indicates there are also important differences. To survive cryogenic temperatures, cells must be permeable to both water and cryoprotective solutes. Neither Drosophila nor Anopheles embryos meet this requirement. Drosophila and Anopheles can both be rendered permeable by exposure to alcohol and alkanes. Although innocuous to the former, the treatment is unfortunately lethal to the latter because, paradoxically, their water permeability is too high. We now, however, have achieved survival of permeabilized young Anopheles embryos by substituting air-drying for alcohol. A second serious problem with both Drosophila and Anopheles is that they exhibit high sensitivity to chilling even in the absence of freezing. High sensitivity to chilling forces one to cryopreservation by the more demanding vitrification approach. Older Anopheles embryos are much less chill sensitive but they are refractory to the permeabilization procedures effective in younger embryos. We propose approaches to circumventing the chill sensitivity of younger embryos and we propose a number of sequential physical and chemical approaches to permeabilizing older embryos. The physical approaches include bombardment with alpha particles and electroporation. The chemical approaches include extraction with supercritical CO2.