非常感谢西尔克咸鱼，莎拉·基弗，迈克尔·韦斯的Conny BARTSCH和西尔克福格特 - Heucke在现场热烈的样本采集，以Janett比肯费尔德为继续提供援助，以乌拉Kobalz为美丽的凝胶和托比亚斯克劳斯的道义上的支持。

所有的程序均符合动物保护（TierSchG）德国法律执行。 试剂和消耗品的1。准备<ol><li>准备在分子级水5％（W / W）CHELEX-100的解决方案。准备200μl，以标准的1.5毫升的反应管等分。由于CHELEX树脂沉淀快起停牌，有必要重新同质化的不断暂停等分试样的制备过程中。这是可取的制备50毫升或多个在一个批次中，并保持该悬浮液用磁力搅拌器均质化。该CHELEX溶液可保存几年在环境条件下。 </li><li>切的Whatman滤纸片，其宽度比鸟要测试的喙宽度小。纸张的长度取决于该方法采取该示例： <ol><li>使用镊子，将片应该足够长，以使样品收集而不被插入到鸟喙的钳子。作为粗略估计，为0.5厘米的长喙一张纸用1厘米的长度应该罚款。 </li><li>如果不使用镊子，这块一定要长，但片的刚度还是应该让上皮细胞取样。 </li></ol></li><li>制备PCR预混物的一个等份对每个样品进行分析。对于PCR中的25的总体积微升的混合物含有0.18毫的dNTPs，2毫摩尔MgCl 2，70 mM的Tris-盐酸，17.25毫硫酸铵，0.1％吐温-20，0.32μMP2引物（TCTGCATCGCTAAATCCTTT），0.32μMP8底漆（CTCCCAAGGATGAGRAAYTG）16和2个单位的Taq聚合酶。自制Taq聚合酶17都可以使用。以允许另外的DNA的溶液的最大量的，6微升预混物可以制备并储存于-20℃下几个星期。 </li></ol> 2，样品采集戴手套是不是鸟类性别决定的PCR引物不能退火到人类的DNA是必不可少的。为其它基因分型的目的可能是可取的。 <ol><li>捕获感兴趣的鸟，轻轻地握在一只手如用&#39;铃声的抓地力&#39;18。请务必不要挤鸟。 </li><li>持有一张Whatman滤纸用钳子和刷几次穿过颊，舌片和后鼻孔的内侧。一般样品少于30秒内获得的。 <ol><li>成年动物：根据种类的不同，可能很难拿到鸟来打开它的嘴。平时接触喙的侧面和应用温和的压力会奏效。 </li><li>雏鸟：从刚孵化的雏鸟或采样是特别容易使用这种技术，因为他们的乞讨行为提供了方便地访问到嘴的内部。它甚至有可能得到的样品没有处理它们，因为它们很容易乞求例如 ，当他们的巢箱被打开。 </li></ol></li><li>紧随纸张存放在一个准备好的反应管里包含宁200微升的5％（W / W）CHELEX-100溶液。 </li></ol>如果您还打算/需要标记的小鸡，现在这样做是在第6节。 3，储存样本的DNA提取前<ol><li>如果在实验室工作，店内样品于-20°C。如果这是不可能的或困难的（ 例如，在现场）店内样品在环境温度下。 </li><li>样品可在温度范围从室温到-20℃下存放3年以上</li></ol> 4，DNA的提取<ol><li>如果样品被冻结，解冻它在室温下。 </li><li>确保纸是在浸没在CHELEX-100溶液的试管底部。 </li><li>孵育样品15分钟，在56℃下</li><li>短暂涡旋后短暂离心管的内容。 </li><li>孵育样品为8分钟，在100℃下</li><li>旋转样品以15,000 xg离心3分钟。 </li><li>使用supernatant为后续的基因分型。 </li></ol> 5。分子性别鉴定<ol><li>准备一台PCR管，每样6微升预混料加为负（强制性）和阳性对照（可选）两个额外的管子。对于常规的性别决定包括从最后一次成功的绝育作为阳性对照样品。 </li><li>从提取DNA加19微升上清到PCR预混物。确保从溶液的表面到吸管以避免CHELEX珠残留。这是至关重要的，因为CHELEX是阳离子交换，从而严重地抑制所有的酶促反应。 </li><li>运行以下的PCR程序：培养在94℃，3分钟，每45个循环，30秒熔化工序94℃，接着是30秒的退火步骤，在55℃，接着在72℃45秒延伸步骤C.在最后的追关闭步骤中，反应在72℃下温育5分钟</li><li>准备一个2％的标准TBE或TAE琼胶<html>本身凝胶来分离PCR产物。 </li><li>运行适当的电压设置的琼脂糖凝胶。 </li><li>可视化的乐队在紫外光下，拍摄一张照片。确保从始发女性样本中的两个频段是完全分离。 </li><li>由一个（男）或两个（女）乐队的存在从女性样本区分男性。 </li></ol> 6。标记雏鸟<ol><li>检查巢刚孵出的小鸡按计划适当的品种/学习（ 如日常检查，在早晨被告知因为大多数小鸡孵化即可）。 </li><li>剪下每个鸡的羽毛巢内的不同特征的位置。在斑胸草雀起伏塔夫茨增长跨越雏鸟的身体（ 图4A）四个特征和不同的领域。为每个小鸡切一个区域（ 图4B）。如果有在一个巢四个以上的小鸡，独特的4&#39;发型&#39;可以组合使用。 </li></ol>ove_content“&gt;对斑胸草雀：适用腿带十天后孵化，当羽毛变得很难检测和雏鸟尺寸允许振铃。

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+buccal+swabs+for+sampling+DNA+from+nestling+and+adult+birds.">Use of buccal swabs for sampling DNA from nestling and adult birds.</a> Wildl. Soc. Bull. 34, 1094-1100 (2006).</li><li>Brubaker, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+noninvasive,+direct+real-time+PCR+method+for+sex+determination+in+multiple+avian+species.">A noninvasive, direct real-time PCR method for sex determination in multiple avian species.</a> Mol. Ecol. Resour. 11, 415-417 (2011).</li><li>Arima, H., Ohnishi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+avian+buccal+cells+for+molecular+sexing.">Usefulness of avian buccal cells for molecular sexing.</a> Ornithological Science. 5, 139-143 (2006).</li><li>Seki, S. -. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+sexing+of+individual+Ryukyu+Robins+Erithacus+komadori+using+buccal+cells+as+a+non-invasive+source+of+DNA.">Molecular sexing of individual Ryukyu Robins Erithacus komadori using buccal cells as a non-invasive source of DNA.</a> Ornithological Science. 2, 135-137 (2003).</li><li>Wellbrock, A. H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buccal+swabs+as+a+reliable+source+of+DNA+for+sexing+young+and+adult+Common+Swifts+(Apus+apus).">Buccal swabs as a reliable source of DNA for sexing young and adult Common Swifts (Apus apus).</a> J. Ornithol. 153, 991-994 (2012).</li><li>Marion, W. R., Shamis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annotated-Bibliography+of+Bird+Marking+Techniques.">Annotated-Bibliography of Bird Marking Techniques.</a> Bird Banding. 48, 42-61 (1977).</li><li>Scharff, C., Adam, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenetics+of+birdsong.">Neurogenetics of birdsong.</a> Curr. Opin. Neurobiol. , (2012).</li><li>Griffith, S. C., Buchanan, K. 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S., Scharff, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+songbird+as+a+model+for+the+generation+and+learning+of+complex+sequential+behaviors.">The songbird as a model for the generation and learning of complex sequential behaviors.</a> Ilar J. 51, 362-377 (2010).</li><li>Honarmand, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stressful+dieting:+nutritional+conditions+but+not+compensatory+growth+elevate+corticosterone+levels+in+zebra+finch+nestlings+and+fledglings.">Stressful dieting: nutritional conditions but not compensatory growth elevate corticosterone levels in zebra finch nestlings and fledglings.</a> PLoS On. 5, (1371).</li><li>Krause, E. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebra+finch+nestlings+beg+more+under+better+nutritional+conditions.">Zebra finch nestlings beg more under better nutritional conditions.</a> Behaviour. 148, 1239-1255 (2011).</li><li>Griffiths, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+test+to+sex+most+birds.">A DNA test to sex most birds.</a> Mol. Ecol. 7, 1071-1075 (1998).</li><li>Pluthero, F. 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作者什么都没有透露。

使用CHELEX口腔拭子性别鉴定表现出非常高的成功率。切割幼体羽绒羽毛启用雏鸟之间的差异，直到腿带是可能的。 从口腔拭子CHELEX-DNA的提取产生足够的DNA，成功地进行分子性别鉴定。性别决定是100％正确的，因为验证的性别二态性的羽毛。这里报道的成功率明显高于报道的前两个研究7,9率较高。该DNA提取与CHELEX最大限度地减少它的原料可能会丢失移液和沉淀步骤。?...

个体识别，性别鉴定和基因分型是在各种实验研究的基本先决条件。获得的DNA轴承材料和标识对象明确（即使在早期的年龄）应该有生理，行为和生存的影响微乎其微。只要有可能，侵入性操作应根据存款准备金率原则1是可以避免的。 非侵入性的方法不仅有利于动物，但也可能会提高所获得的数据作为动物较少受到处理。 在鸟类中，DNA绝育可以对一些非侵入性地获得的材料如粪便2，羽毛3,4或口腔拭子3,5,9进行。无论拍摄对象的状况和年龄口腔拭子是禽流性别鉴定方法的选择，因为它们很容易进行，很少不和处理是短暂的。 到目前为止，从口腔拭子DNA用市售试剂盒3,6或耗时标准的DNA提取方案3,6-8中任一萃取。工具包不仅是相当昂贵的，但他们的协议可以并处挑战野外作业。一些程序细节， 如干燥和样品的培养，是不实际的领域。特别是在实验方案需要性取决于治疗从早在几分钟后的阴影的设定，有督促进行快速，非侵入性，可靠和容易的方法得到的结果。 在整个禽流类群相当工具箱标志着个人已经发展10。可用技术的广泛约占各种研究目标，物种和预算。然而，标志着小雏鸟已面临研究人员更多的挑战。在一些物种（ 如燕雀）雏鸟太小，适用腿带和需要替代方法，这不改变亲子行为。由于意识和改善动物福利和实地和实验室研究技术的兴趣不断增长，使用非侵入性技术被大力鼓励和首选。 该协议提供了一种非侵入性，快速，易于辨认和持久的方法运用腿带之前，单独标记非常年轻的雏鸟是可行的。此标记方法介绍了一个最重要的禽流感的实验室模型的物种，斑胸草雀（Taeniopygia弄蝶 ）11-13。该协议符合所有的个人标记技术对10先前公布的目标，并已经被成功地应用于14,15。

<table border="0" cellpadding="0" cellspacing="0" width="702">
	<tbody>
		<tr height="75">
			<td height="75" style="height:75px;width:191px;">Whatman 3MM Chromatography paper</td>
			<td style="width:163px;">e.g. Fisher Scientific</td>
			<td style="width:269px;">No.:3030-153</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:191px;">Chelex 100 Resin</td>
			<td style="width:163px;">Biorad</td>
			<td style="width:269px;">#143-2832</td>
			<td></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:191px;">Taq polymerase</td>
			<td style="width:163px;">addgene</td>
			<td style="width:269px;">http://www.addgene.org/25712/</td>
			<td></td>
		</tr>
		<tr height="75">
			<td height="75" style="height:75px;width:191px;">leg band (Flexi number rings for birds, diameter 2,5 mm)</td>
			<td style="width:163px;">Horst Stengel &#38; Sohn</td>
			<td style="width:269px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

who is who? non-invasive methods to individually sex and mark altricial chicks

许多实验需要及早确定后代的性别以及早期新生儿标记为个人的认可。根据动物福利指导方针​​，非侵入性技术，应首选（倘适用）。在我们的团队，我们工作在不同的曲种鸟类在实验室和现场，而我们成功地应用非侵入性的方法，以性别和单独标记小鸡。本文提出了一种综合性的非侵入性的工具盒。绝育鸟类之前第二性性状的表达需要DNA的轴承材料用于PCR的集合。我们通过提取来自口腔拭子DNA建立了一个快速简便的方法对任何年龄（孵化后）的性鸟类。结果可以在3小时内获得。对于个别标志着小鸡羽绒羽毛被修剪的具体模式允许孵化秩序内快速识别。这套方法是简单地适用于配备标准实验室和特别适合干活在该领域，因为没有专用设备克所需的采样和存储。处理雏鸡的最小化和标记和性别鉴定技术是无创性，从而支持动物福利指导方针​​的存款准备金率原则。

<html>口腔拭子可以用来提取DNA进行性别鉴定中的各种小鸟的 ，加那利群岛（Serinus加那利岛 ），孟加拉雀（ 白腰文鸟芨 ），南丁格尔（ 红点megarhynchos），大山雀（ 大山雀 ）和黑鹂（ 鸫 -样品采自斑胸草雀（5年Taeniopygia雀，99个人，年龄零天）收集merula）（所有其他物种：样本大小1-3，年龄不详）（ 图1）。 <p class...

Watch this Scientific Journal Video about Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks at JoVE.com

Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

该协议提供了一套便捷的方法，使极为快速，简便，无创，可靠，成本低，分子性别鉴定鸟类和它们的无创，快速，安全，易于辨认孵化后不久标记。仅限雏鸡的处理是必需的。的方法，这种方便的工具箱完全符合RRR-指引。

谁是谁？非侵入性的方法，以个别性和马克晚成小鸡

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal ...

who-is-who-non-invasive-methods-to-individually-sex-mark-altricial

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JoVE Journal

Biology

18.2K 次观看.  Freie Universität Berlin. 该协议提供了一套便捷的方法，使极为快速，简便，无创，可靠，成本低，分子性别鉴定鸟类和它们的无创，快速，安全，易于辨认孵化后不久标记。仅限雏鸡的处理是必需的。的方法，这种方便的工具箱完全符合RRR-指引。 

视频: Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

科研

生物学

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

非侵入性的三维可视化与亚微米分辨率使用同步加速器X射线断层扫描

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

一个磁性的系绳系统，以调查在果蝇的视觉和嗅觉介导的飞行控制

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually scattered cells and small cell groups. This technique uses HistoGel (Thermo Scientific) with conventional laboratory equipment.
The use of cell block sections is a valuable ancillary tool for evaluation of non-gynecologic cytology. They enable the cytopathologist to study additional morphologic specimen detail including the architecture of the lesion. Most importantly, they allow for the evaluation of ancillary studies such as immunocytochemistry, in-situ hybridization tests (FISH/CISH) and in-situ polymerase chain reaction (PCR). Traditional cell block preparation techniques have mostly been applied to non-gynecologic cytology specimens, typically for body fluid effusions and fine needle aspiration biopsies.
Liquid based cervicovaginal specimens are relatively less cellular than their non-gynecologic counterparts with many individual scattered cells. Because of this, adequate cellularity within the cell block sections is difficult to achieve. In addition, the histotechnologist sectioning the block cannot visualize the level at which the cells are at the highest concentration. Therefore, it is difficult to monitor the appropriate level at which sections can be selected to be transferred to the glass slides for testing. As a result, the area of the cell block with the cells of interest may be missed, either by cutting past or not cutting deep enough. Current protocol for Shidham's method addresses these issues. Although this protocol is standardized and reported for gynecologic liquid based cytology specimens, it can also be applied to non-gynecologic specimens such as effusion fluids, FNA, brushings, cyst contents etc for improved quality of diagnostic material in cell block sections.

Shidham's method for preparation of cell blocks with AV-marker from cytology specimens containing individually scattered cells and small cell groups.

个别细胞散为主细胞学标本的细胞块的制备

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

一个实用的方法遗传诱导的命运映射：一个视觉引导标记和跟踪细胞在体内</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

方法发现小鼠BCRP1的另类用法子和转录调控

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

将细胞质Foci分类为哺乳动物应激颗粒的方法

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...