泄殖腔按摩与精子 DNA 分离法采集禽精
Summary
本协议描述泄殖腔按摩, 一种非侵入性的手工技术, 用于从禽类中收集精子, 以及使用商业提取试剂盒进行 DNA 提取, 并进行修改。
Abstract
收集的精子可能是有用的广泛的应用, 包括研究涉及精质, 精子端粒动力学和表观遗传学。鸟类是生物学研究中广泛使用的学科, 是涉及反复精子样本研究的理想选择。然而, 对于那些希望学习如何从禽类精子中收集和提取 DNA 的人来说, 目前还没有多少资源可用。在这里, 我们描述泄殖腔按摩, 一个温和的, 非侵入性的手工技术收集禽类精子。虽然这项技术是建立在文献, 它可能是很难从可用的描述学习。我们还提供信息, 从禽精提取 DNA 的商业提取试剂盒的修改。泄殖腔按摩可以很容易地用于任何小到中型雄鸟的生殖条件。在收集后, 可以立即使用精子进行运动化验, 或在本协议所述的 DNA 提取后冷冻。这一提取协议被提炼为禽精, 并已成功地用于收集的样本从几个雀物种 (过路人幼雏, Spizella passerina, Haemorhous mexicanus, 和鸫 migratorius) 和一个 columbid (鸽)。
Introduction
鸟类是研究精子质量和竞争的理想科目1, 精子端粒动力学2, 遗传学的3, 和类似的主题, 因为它们广泛用于生物研究和精子可以很容易地取样使用泄殖腔按摩.泄殖腔按摩是一种温和的, 非侵入性的手工收集精子的技术, 从鸟类4,5,6。重复样品可以很容易获得, 不需要特殊的工具, 使其易于在现场或实验室执行。虽然泄殖腔按摩已经使用了几十年, 很难从现有的书面说明。本刊物旨在减少学习泄殖腔按摩所涉及的时间和不确定因素。
从鸟类收集的精子使用泄殖腔按摩可立即用于运动评估4,7或人工授精8, 或冷冻其他用途, 如高级成像和 DNA 提取。来自雀鸟的精子样本很小, 但含有浓密的精子。DNA 的提取使用一个商业提取试剂盒简单, 与修改, 以克服专门的保护功能的精子9。提取后, 精子端粒可以使用 qPCR10进行测量。
Protocol
Representative Results
Discussion
本文介绍了一种简单可靠的生殖活性条件下从中小鸟类采集精子的方法, 并从禽精中提取 DNA。
所描述的精子 DNA 提取协议是从一个简单的套件修改, 但被提炼为使用在禽类的精子。精子抗裂解的标准萃取化学品9,13, 从形收集的精样本是相当小的 (通常是少数升)。这些因素提出了在65° c 的情况下使用适当浓度的德勤和孵化成功克服的?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢何塞诺格拉的早期协助泄殖腔按摩, 和杰弗里 Kittilson 的实验室支持。这项工作得到了 EPSCoR FAR0022429 奖对 BJH 的支持。出版费用得到了 NDSU 生物科学部的慷慨支持。
Materials
| Microhematocrit capillary tubes | Fisherbrand | 22-362566 | Any supplier can be used. Smaller tubes may be more effective for collection of small semen samples, such as from very small passerine birds like chipping sparrow. |
| Microcentrifuge tubes | Any | ||
| 1x PBS | Any | Used for storing semen samples prior to DNA extraction. | |
| DL-Dithiothreitol | Sigma-Aldrich | D0632 | |
| Tris base | Sigma-Aldrich | 10708976001 | Any supplier can be used. |
| EDTA | Sigma-Aldrich | 1233508 | Any supplier can be used. |
| NaCl | Sigma-Aldrich | 793566 | Any supplier can be used. |
| SDS | Sigma-Aldrich | 1614363 | Any supplier can be used. |
| QIAamp DNA micro kit | Qiagen | 56304 | Qiagen QIAamp DNA micro kit is recommended specifically. This DNA extraction protocol has been attempted with other commercial kits but with little success. The reasons for this are unknown to us. |
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