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。

该议定书涉及脊椎动物的主题, 并已批准的机构动物保育和使用委员会 (IACUC) 在北达科他州立大学。
1. 使用泄殖腔按摩的雀鸟的精子收集
注: 泄殖腔按摩是一种有效的生殖活禽的精子收集技术, 但可以在适当的圈养情况下成功地在繁殖季节之外进行。在使用泄殖腔按摩之前, 应确定目标物种的生殖活动。这种技术可以用于野生鸟类捕获或圈养鸟类。
<ol><li>从雄鸟开始, (在繁殖条件) 達的握在非主导的手, 转移鸟到支配的手与它的腹侧接触手掌 (图 1)。<ol>
<li>确保鸟的头部离手掌的侧边最近。用小指、环和中指轻轻地保护头部和身体。这就使得鸟的排气口和尾部露出来了。</li>
		<li>用手掌把鸟的腿松开或轻轻地克制。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55324/55324fig1.jpg"> 图 1: 泄殖腔按摩手柄.这只鸟在占优势的手上被轻轻地固定着, 它的腹侧接触手掌, 头部被小指固定。<a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="2"><li>将占主导地位的食指和拇指放在泄殖腔的任一侧, 轻轻地捏住手指, 对尾部施加非常轻微的压力 (图 2)。泄殖腔将埃弗特, 露出泄殖腔粘膜 (粉红色内部)。 注意: 如果鸟目前没有生产精子, 泄殖腔突起可以小, 泄殖腔很难埃弗特。有经验, 很容易区分 cloacas 的生殖活动和非活动的男性5,11。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55324/55324fig2.jpg"> 图 2: 手指位置和泄殖腔突起.埃弗特泄殖腔通过施加轻微的压力到上级端 (A)。在生殖活跃的雄性雀中, 泄殖腔突起明显且牢固 (B)。<a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="3"><li>用非显性手, 将食指的垫放在尾巴底部的背侧以稳定手, 然后将拇指的尖端放在外翻泄殖腔的下方。 注意: 短而平滑的缩略图有利于进行泄殖腔按摩, 保护鸟免受意外伤害。</li>
	<li>移动非显性拇指深和 cranially 在一个重复的运动中压, 保持低于泄殖腔。继续, 直到鸟射精。根据个人, 这通常需要在1和几十个中风。</li>
	<li>鸟一射精, 就在 microhematocrit 管中收集精子。重复泄殖腔按摩以获得多射精。 注: 雀的精子呈浅褐色, 通常有较厚的稠度。从泄殖腔中排出的白色或深色物质不是精子, 可能会污染精子样本。在泄殖腔或与精子混合时出现少量的血液, 通常是由于按摩时压力太大造成的。</li>
	<li>对于 DNA 提取, 将精子放入离心管中, 20 µL 1x PBS (pH 7.4)。注: 1x PBS 中贮存的精子不适合人工受精, 但在-80 ° c 贮存时可用于 DNA 提取。PBS 可以是室温或冷冻的, 但在储存精子样本时应处于液态状态。<ol>
<li>通过在复合显微镜下观察少量的稀释样品 (图 3), 确认样品中是否存在精子。</li>
	</ol>
</li>
	<li>将稀释的精子贮存在-80 ° c 直至提取。</li>
</ol>2. 从禽精中提取 DNA
注: 本协议已通过多个商业 dna 提取试剂盒进行了测试, 但仅在使用一个 dna 套件12时才成功。从套件协议中修改的步骤由 * 表示。
<ol><li>在水中准备一个1米的糖 (德勤) 溶液, 并使用涡流混合。<ol>
<li>分的解决方案, 并存储在-20 ° c, 直到使用前立即。每个样品要提取需要10µL 1 M 的解决方案。</li>
	</ol>
</li>
	<li>准备一个包含20毫米三氯的缓冲液, pH 值 8.0;20毫米 EDTA;200毫米氯化钠;和 4% SDS。要提取的每个样本需要此缓冲区的90µL。<ol>
<li>在室温下存储缓冲区。如果它已经沉淀, 温暖它到56° c 溶化。</li>
	</ol>
</li>
	<li>将装有摇杆或振动筛的恒温箱预热至65° c。</li>
	<li>在开始提取之前, 在步骤2.2 中为每个抽取的样本准备10µL 的90µL 的缓冲液, 并在提取100样本时,例如900 µL 缓冲器加10µL 的解决方案。这种混合物将被称为 "数码缓冲"。</li>
	<li>添加70µL 1x PBS 到整个稀释的精子样本和混合良好使用涡流。</li>
	<li>添加100µL 的数码缓冲和10µL 蛋白酶 K 溶液的样品。漩涡20秒, 然后孵化在预热孵化器1小时的摇杆或振动筛。</li>
	<li>添加200µL 缓冲 AL 和200µL 新鲜乙醇 (100%) 和漩涡20秒。</li>
	<li>将整个样品转移到旋转柱, 离心机1分钟, 6000 x g. 丢弃收集管。</li>
	<li>将列放在新管上, 并添加500µL 准备的缓冲 AW1。离心机1分钟在 6000 x g. 丢弃收集管。</li>
	<li>将列放在新管上, 并添加500µL 准备的缓冲 AW2。离心机1分钟, 在 6000 x g. 丢弃流, 并将列放回管上。</li>
	<li>离心机3分钟在 2万 x g. 丢弃收集管, 将柱放在1.5 毫升离心管上。</li>
	<li>吸管35µL 将 AE 缓冲器直接放在柱膜上, 室温孵育5分钟。</li>
	<li>离心机1分钟在 1.4万 x g. 丢弃自旋柱, 并在-80 ° c 处储存提取的样品。</li>
</ol>

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作者声明他们没有竞争的金融利益。

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?...

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。

<table border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microhematocrit capillary tubes</td>
			<td style="width:71px;">Fisherbrand</td>
			<td style="width:119px;">22-362566</td>
			<td style="width:167px;">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.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tubes</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1x PBS</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td>Used for storing semen samples prior to DNA extraction.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DL-Dithiothreitol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris base</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">10708976001</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EDTA</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">1233508</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">793566</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SDS</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">1614363</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAamp DNA micro kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">56304</td>
			<td>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.&#160;</td>
		</tr>
	</tbody>
</table>

avian semen collection by cloacal massage and isolation of dna from sperm

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)。

泄殖腔按摩和 DNA 提取使用所描述的协议已经对几个雀物种和一个 columbid (表 1) 进行。在泄殖腔按摩收集的精子样本中, 通过在400X 放大倍数显微镜下观察少量稀释后的样品 (图 3), 证实了精的存在。从家庭麻雀收集的精子样本中提取的 dna 使用这里所描述的协议, 八样品在凝胶电泳和 dna 阶梯上运行 (三样本可以在图 4...

Watch this Scientific Journal Video about Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm at JoVE.com

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

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。

泄殖腔按摩与精子 DNA 分离法采集禽精

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

avian-semen-collection-cloacal-massage-isolation-dna-from

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Biology

15.8K 次观看.  North Dakota State University. 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。

视频: Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. 

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female&#x2019;s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

收获的小鼠精子和人工授精

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

科研

生物学

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

人类胰岛的分离：第一部分：收集和胰腺组织消化

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

禽流感胚胎瓣膜祖细胞的分离和培养

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

一种实用的新的方法来提取基因组DNA保存血浆蛋白从血液采集套件

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

从环境中自然产生的趋磁细菌的采集，分离和富集

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

子宫 - 输卵管胚胎移植和输精管结扎术在小鼠模型

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

培养<em&gt;秀丽隐杆线虫</em &gt;在无菌液体介质与创造转基因蠕虫通过微粒轰击

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA电穿孔，肌纤维的分离和成像

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

泄殖腔按摩与精子 DNA 分离法采集禽精

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