Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes.
Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes.
复制标题
DOI：
10.3791/55378
复制
发表时间：
2017-11-22
期刊：
影响因子：
--
通讯作者：
Berkowitz KM
中科院分区：
文献类型：
--
作者：
Dia F;Strange T;Liang J;Hamilton J;Berkowitz KM
减数分裂是配子通过一轮DNA复制和连续两轮染色体分离形成的发育过程。哺乳动物减数分裂可以通过利用制备减数分裂染色体铺展的技术来检查。在这里，我们展示了一种方法，从小鼠精母细胞制备表面扩散核。哺乳动物减数分裂是发生在生殖细胞中的动态发育过程，可以进行研究和表征。使用一种方法来传播细胞核的表面上的幻灯片（而不是从高处下降），我们展示了一个优化的技术，在1997年首次描述的小鼠精母细胞。这种方法被广泛用于实验室研究哺乳动物减数分裂，因为它产生了大量的高质量的细胞核，这些细胞核经历了前期I的亚阶段。首先将生精小管置于低渗溶液中以使精母细胞膨胀。然后将精母细胞释放到蔗糖溶液中以产生细胞悬浮液，并将细胞核扩散到固定剂浸泡的载玻片上。免疫染色后，可以检查与减数分裂过程密切相关的蛋白质的多样性。例如，联会复合体的蛋白质，一种将同源物的染色体轴/核心连接在一起的三方结构，可以很容易地可视化。减数分裂重组蛋白通过同源重组参与DNA双链断裂的修复，也可以通过免疫染色来评估前期I的进展。在这里，我们详细描述和演示了一种广泛用于研究哺乳动物精母细胞减数分裂的技术，从幼年或成年雄性小鼠。
Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes. Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.
登录
查看更多内容
DOI：
10.1111/febs.13301
发表时间：
2015-07
期刊：
The FEBS journal
影响因子：
--
作者：
Rankin S
通讯作者：
Rankin S
DOI：
10.1007/978-1-60761-103-5_17
发表时间：
2009-01-01
期刊：
MEIOSIS, VOL 2: CYTOLOGICAL METHODS
影响因子：
--
作者：
La Salle, Sophie;Sun, Fengyun;Handel, Mary Ann
通讯作者：
Handel, Mary Ann
影响因子：
4.5
作者：
Berkowitz KM;Sowash AR;Koenig LR;Urcuyo D;Khan F;Yang F;Wang PJ;Jongens TA;Kaestner KH
通讯作者：
Kaestner KH
DOI：
10.1007/978-1-4939-0888-2_11
发表时间：
2014
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Brooker, Amanda S;Berkowitz, Karen M
通讯作者：
Berkowitz, Karen M
影响因子：
1.6
作者：
ASHLEY, T;PLUG, AW;WARD, DC
通讯作者：
WARD, DC