Tsuchiya Dai, Eads Brian D, Zolan Miriam E
Department of Biology, Indiana University, Bloomington, IN, USA.
Methods Mol Biol. 2009;558:235-49. doi: 10.1007/978-1-60761-103-5_14.
The genus Daphnia has an intriguing reproductive mode of cyclical parthenogenesis. This reproductive mode has been studied for centuries, but cytogenetic information is lacking due to technical limitations of classical methods. We have developed methods for the preparation and examination of meiotic chromosomes of Daphnia pulex from oocytes and spermatocytes. Oocyte chromosome preparations are obtained by isolating individual oocytes after the release of yolk granules from the ovary using pressure and capillary action. Spermatocyte chromosomes are prepared using a conventional squash method. Cryosectioning is an easy and fast way to prepare sections. We also illustrate the application of immunofluorescence staining against alpha tubulin, as well as fluorescence in situ hybridization (FISH) using the intergenic spacer of ribosomal DNA or single-copy cosmid clones.
水蚤属具有一种有趣的周期性孤雌生殖繁殖模式。这种繁殖模式已经被研究了几个世纪，但由于传统方法的技术限制，细胞遗传学信息仍然缺乏。我们已经开发出了从卵母细胞和精母细胞中制备和检测蚤状溞减数分裂染色体的方法。卵母细胞染色体的制备是通过使用压力和毛细作用从卵巢释放卵黄颗粒后分离单个卵母细胞来实现的。精母细胞染色体则采用传统的压片法制备。冰冻切片是一种简单快速的切片制备方法。我们还展示了抗α微管蛋白免疫荧光染色的应用，以及使用核糖体DNA基因间隔区或单拷贝黏粒克隆进行荧光原位杂交（FISH）。
