减数分裂纺锤体评估在小鼠卵母细胞的siRNA介导沉默
Summary
在这里，我们提出了一个协议，用于特定的siRNA介导的mRNA枯竭其次是免疫荧光分析来评估减数分裂纺锤体组装和组织在小鼠卵母细胞。这个协议是适合体外转录耗尽的和不同的主轴和/或微管组织中心相关因子中的卵母细胞的功能评价。
Abstract
Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down’s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.
Introduction
减数分裂是发生在配子（卵子和精子），涉及两个连续的部门不干预DNA合成减数分裂我和减数分裂-II， 分别为1时分离的同源染色体和姊妹染色单体独特的分裂过程。在卵母细胞在减数分裂中染色体分离错误可能导致非整倍体，它是由胚胎在受精过程中继承。值得注意的是，非整倍体在发育中的胚胎的发生率的增加而前进产妇年龄和是先天性出生缺陷的主要原因，以及在妇女妊娠1,2-损失，因此，强调理解非整倍体的过程中减数分裂的分子基础的重要需求。
在细胞分裂，染色体分离是关键取决于微管主轴装置，并建立稳定的染色体微管相互作用进行正确连接到对面纺锤体组装极。重要的是，减数分裂纺锤体形成在哺乳动物卵母细胞不同于有丝分裂中的体细胞，并通过独特的微管组织中心（MTOCs）缺乏中心粒3,4-调节。必要的微核和组织必需的蛋白质定位于卵母细胞MTOCs，包括γ – 微管蛋白的催化微管组装。另外，粒周功能作为必要支架蛋白，其结合和锚γ微管蛋白以及其他因素在MTOCs 5。值得注意的是，我们的研究表明，主要的微管组织中心相关蛋白的耗尽扰乱减数分裂纺锤体组织，并导致在卵母细胞的染色体分离的错误，这是不充分的主轴组件的检查点（SAC）6,7-解决。因此，在主轴的稳定性的缺陷，即不会触发减数分裂阻滞，姿势在促进非整倍体一个显著风险。尽管在纺锤体组装和组织下，卵母细胞微管组织中心PROT的重要作用EIN组成和功能仍然知之甚少。
测试特定的目标蛋白质的功能在哺乳动物卵母细胞是具有挑战性的，因为细胞变得转录静止恢复减数分裂8,9前不久。因此，排卵前的卵母细胞依靠产妇基因店恢复减数分裂和支持减数分裂和受精10,11后的第一次卵裂的分裂。 RNA干扰（RNAi）介导的mRNA转录物在哺乳动物卵母细胞退化的功效是公认的，并在减数分裂成熟招募翻译母系RNA是特别适合于siRNA的靶向12-14中 。因此，短干扰RNA显微注射（的siRNA）为卵母细胞提供了一个有价值的方法来耗尽目标基因进行功能测试。
在这里，我们描述了小鼠卵母细胞和具体的transcri siRNA-介导的耗尽的分离方法点测试的必要微管组织中心相关蛋白的功能，粒周。此外，我们描述免疫荧光分析条件来评估减数分裂纺锤体形成在卵母细胞。
Protocol
Representative Results
Discussion
尽管有多种方法外源核酸转移进入体细胞，例如电穿孔和转染，显微注射是用于递送RNA分子的转录成静止小鼠卵母细胞的最佳方法。目前的协议提供了一种有效的方法用于体外耗尽特异mRNAs，使不同的主轴和/或微管组织中心相关因子中的卵母细胞的功能测试。这种做法会导致效率的成绩单枯竭和高度的适应性。虽然的siRNA被用于特异性靶向PCNT转录，类似的条件，可以采用靶向感兴趣?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was supported in part by the University of Georgia, and a grant (HD071330) from the National Institutes of Health to MMV.
Materials
| Reagents | |||
| Pregnant Mare's Serum Gonadotropin (PMSG) | EMD Biosciences | 367222 | |
| Minimal Essential Medium (MEM) | *Recipe outlined in Table 1 | ||
| Earle's Balanced Salt Solution (10x) | Sigma | E-7510 | |
| Sodium Bicarbonate | Sigma | S-5761 | |
| Pyruvic Acid, sodium salt | Sigma | P-5280 | |
| Penicillin G, potassium salt | Sigma | P-7794 | |
| Streptomycin Sulfate | Sigma | S-9137 | |
| L-Glutamine | Sigma | G-8540 | |
| EDTA, disodium salt dihydrate | Sigma | E-4884 | |
| Essential Amino Acids (50x) | Gibco | 11130-051 | |
| MEM Vitamin Mixture (100x) | Sigma | M-6895 | |
| Phenol Red solution | Sigma | P-0290 | |
| Bovine Serum Albumin (BSA) | Sigma | A1470 | |
| Milrinone | Sigma | M4659 | |
| Fetal Bovine Serum (FBS) | Hyclone | SH30070.01 | |
| EmbryoMax M2 Media with Hepes | EMD Millipore | MR-015-D | |
| siRNAs targeting Pericentrin | Qiagen | GS18541 | |
| Negative control siRNAs | Qiagen | SI03650318 | |
| Paraformaldehyde (16% solution) | Electron Microscopy Sciences | 15710 | |
| Triton-X | Sigma | T-8787 | |
| Phosphate Buffered Saline (PBS) | Hyclone | SH30028.02 | |
| Anti-Pericentrin (rabbit) | Covance | PRB-432C | |
| Anti-acetylated a-tubulin (mouse) | Sigma | T-6793 | |
| Goat anti-rabbbit Alexa Fluor 488 | Invitrogen | A-21430 | |
| Goat anti -mouse Alexa Fluor 555 | Invitrogen | A-11017 | |
| Major Equipment | |||
| Stereomicroscope (SMZ 800) | Nikon | ||
| Upright Fluorescent Microscope | Leica Microsystems | ||
| Inverted Microscope | Nikon | ||
| Femtojet Micro-injections System | Eppenforf | ||
| Micro manipulators | Eppendorf | ||
| Micro-injection needles (femtotips) | Eppendorf | 930000035 | |
| Holding pipettes (VacuTip) | Eppendorf | 930001015 | |
| Plasticware | |||
| 35mm culture dishes | Corning Life Sciences | 351008 | |
| 4-well plates | Thermo Scientific | 176740 | |
| 96 well plates | Corning Life Sciences | 3367 | |
| 0.45 mm CA Filter System | Corning Life Sciences | 430768 |
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