该协议是基于在实验室研究所生物学物理化学公司，巴黎，法国的弗朗西斯，安德烈沃尔曼学到的技术。作者欠报恩韦芝Bascou，原本星展，以基因工程技术生产的衣，衣在作者实验室的研究是由美国国家科学基金会奖MCB - 0646350的支持。

第1部分：配子株的制备注：所有的操作应在室温下进行。更高或更低的温度会影响受精卵萌发的速度和可能产生不利影响的可行性。 重要的考虑因素：重要的是要交配的菌株是增长强劲，真菌或细菌污染。如果菌株没有经常被转移，通过丰富培养基上应先产生配子。 <ol><li>要交配的菌株转移到抽头板（100 × 15毫米），每盘1-2株。细胞生长在一个约1厘米宽的集中地带。 </li><li>应该继续下去，直到一个强劲增长的细胞一层厚厚的存在，如有必要的中间传输的TAP板的孵化。尚未最近代的菌株可能需要一个通道，共1周，而经常使用的野生型菌株可能只需要几天没有通道。光敏感的突变体，可能需要更多的时间。 </li><li>从TAP板的所有细胞转移到N10的板，这是缺乏氮，将开始诱导配子。转让厚板坯的细胞，就像他们在水龙头上板。这将有利于随后的细胞集合。 N10的3天之后，细胞准备接受配子。将N10的细胞分裂缓慢，但细胞应保持深绿色。可行的受精卵将无法获得任何受污染的菌株应该抛弃。 </li></ol>第2部分：交配<ol><li>每一株转移到50毫升锥形瓶中，并在约2.5毫升无菌蒸馏水悬浮。这是可以做到，无论是用一根铁丝环或无菌棒，最重要的是细胞以及暂停，并在团块。水的确切数量应进行调整，使细胞浓度，如用肉眼来衡量，是对每一株相同。 </li><li>细胞是激动的强光下（1.260μE /米2 /秒 ），2小时，在这段时间内的细胞将成为运动型配子。应检查光显微镜下蠕动。如果配子不善游泳，交配可能是低效的。然而，即使是不好的运动型菌株交配频率下降。 </li><li>接下来，平等卷MT和MT -配子结合成一个单一的50 ml容量瓶中。配子留在强光下（1.260μE /米2 /秒 ），不躁动。 </li><li>在光学显微镜下，交配，可以立即检查。对配子形成迅速，而且会出现快速移动的细胞鞭毛加入。如果交配似乎极为迅速，样品应采取30分钟-1小时内生成的受精卵。如果没有，可以交配检查每一个小时，虽然这是可选的。由于倍，给予最佳的受精卵密度会有所不同十字架之间广泛，最好是在不同时期采取等份混合交配后1小时，2小时，3小时和4小时后相结合的配子，例如。这是通过转让300μL为TAP - 3％DIFCO琼脂60 × 15毫米板样品，每盘3-4点。要小心不要搅动混合交配，因为这可能会破坏交配对。 DIFCO琼脂使用，因为它比售价较低的琼脂较少的污染物，它会更容易看到，在显微镜下的受精卵。 可以留在未使用的混合交配烧瓶中过夜，第二天交配反应的质量将得到明显。交配时是有效的，受精卵将坚持在墙上或烧瓶底部，中期将出现明确。甚至烧瓶中搅拌，将无法驱逐的受精卵。如果没有坚持的混合物时，合理的活力激动玻璃，然后交配是低效或未能成行。 </li><li>分接开关Difco公司板包含交配混合物干燥后在无菌罩，盖半掩，直到任何表面的液体被吸收。板块通常是留在18小时（700℃/米2 /秒）的光，然后在铝箔包裹，因为zygospore成熟只会在黑暗的地方。菌株划线，交配时间和日期写板和箔。板存储5至7天，并可以保持较长，虽然合子萌发效率逐步下降。 </li></ol>第3部分：四分体解剖<ol><li>植物细胞应刮掉干点交配的混合物，如手术刀，用钝的刀片。刀片可用于暴露面积的受精卵，将坚持严格的3％琼脂。这一步通常是在下午进行，使受精卵第二天早晨发芽，但还没有发生第二，营养师收益率8后代。衣生物学家很快就学会认识到，在显微镜下的受精卵，因为它们比营养细胞大，并配备了厚厚zygospore的墙，给出了一个黑色的轮廓。 Zygospores经常出现黄色的，但也可能是有点绿色。最重要的品格是，他们坚持以琼脂，而营养细胞很容易移到一边。然而，如果过于干燥板这会不会是这样。此外，如果交配混合物过于密集，受精卵可能形成一个垫子，这将使转让个人受精卵很难或者不可能。在步骤2.1中建立适当的密度，将很快与经验教训。 </li><li>玻璃针准备为收集受精卵和分离四分后代。这些都是准备拉3毫米的玻璃棒，小火焰，生成一个长而细的的螺纹，可折断。这些锥形目的是平滑的提示或弯曲成钩，在低的火焰（如酒精或燃烧的匹配）的简要加热。准备3个或更多的玻璃针，因为他们很容易地打破，特别是对没有经验的用户。 </li><li>受精卵都聚集在3％的TAP - Difco公司使用的玻璃针成一小堆的板。一个平方米的琼脂（可达0.5厘米2），其中包含的受精卵形成和使用沉闷的手术刀切除。然后，琼脂块放在正面朝下一个15毫米的1.5％TAP Difco公司板，下滑沿板顶部约1.5厘米的水平线，分发的受精卵。顶部的水平线，和一个约1.5厘米，水平和垂直间距0.5厘米的网格蚀刻成板的背面。网格将用于翌日作为一个分离的四分体的后代（网格，也可以一天清扫蚀刻）的指导。最后，单个受精卵移动到每条垂直线顶端的水平线的交叉点，约20-30％合子板。虽然只有12家左右将被解剖，而不是每一个受精卵发芽，有的将划分爆破前的两倍，那么多余的受精卵是必要的。 </li><li>后分发和植物细胞的受精卵，已不慎从3％琼脂平板转移被杀害揭露为30秒，在一个玻璃盘，含薄（1-2毫米）的氯仿层倒板。板和氯仿之间的距离应为5厘米左右。如果使用太多氯仿，如果距离过小或伟大，一个风险要么杀死受精卵或不杀生的营养细胞。 zygospores分布在发芽板，以确保选择性，氯仿治疗必须立即进行。 </li><li>发芽需16-20小时，大多数菌株，但会有所不同基因型与受精卵的年龄。发芽，板放置在弱光下（239℃/米2 /秒），或在中等光线变暗覆盖板，用纸巾。 </li><li>发芽的受精卵与它们内部的女儿细胞肿胀，有时甚至爆裂的细胞显微镜下可以识别。 ，如果zygospore没有破裂，轻轻触摸玻璃针将打破墙，如果发芽发生。轻轻拖动到下面的受精卵的格点的子细胞。这是最容易做的捕获少量的玻璃针的压力从琼脂发布的液体培养基中的细胞，并拖着这个“水坑”针。一个“保险”之一，解剖一个受精卵，而不是一个营养细胞，曾两次被划分，的形式，除了四个子细胞，“皮肤”的zygospore墙将保持不变。 </li><li>解剖后代的成长，在适当条件下可用于后续的分析，直到可见clonies回升。板材应每天监测污染。 </li></ol>注：代营养二倍体不在这里，但主要涉及电镀交配混合介质，将不支持或者父母的压力增长，但将支持二倍体的增长（例如，两个不同的auxotropic标记）。板都保存在光线，以避免形成受精卵。

<ol>
	<li>Harris, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chlamydomonas+Sourcebook:+Introduction+to+Chlamydomonas+and+Its+Laboratory+Use.">The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and Its Laboratory Use.</a> Conf Proc IEEE Eng Med Biol Soc. , 119-273 (2005).</li></ol>

The authors have nothing to disclose.

遗传分析是简单的在衣 ，但它需要特别的技术，以产生配子和独立的四分体后代。与健康株，这些技术很容易掌握。一些突变株，它成为一门艺术，和读者应参考最近出版的衣历史笔记的原始资料和实用提示第1卷。此外，应该指出的是，有许多变化的基本主题，如上所述，从方法诱导配子，受精卵分布和解剖模式。最终，每个研究员认为自己的舒适区，或适应实验室专用协?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CC-125</td>
<td>Mating type plus strain</td>
<td>Chlamydomonas Stock Center</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CC-124</td>
<td>Mating type minus strain</td>
<td>Chlamydomonas Stock Center</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tris-Acetate-Phosphate (TAP) </td>
<td>Growth medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>N10</td>
<td>Growth medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Difco agar</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fisherbrand Stainless-Steel Blades</td>
<td>Tools</td>
<td>Fisher Scientific</td>
<td>08-916-5B</td>
<td>&nbsp;</td>
<tr>
<td>BD Surgical Blade Handles</td>
<td>Tools</td>
<td>Fisher Scientific</td>
<td>08-914-5</td>
<td>&nbsp;</td>
<tr>
<td>Fisherbrand Metal Scalpels</td>
<td>Tools</td>
<td>Fisher Scientific</td>
<td>08-920A</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

mating and tetrad separation of chlamydomonas reinhardtii for genetic analysis

单细胞绿藻<em&gt;衣藻</em&gt;（<em&gt;衣</em&gt;）已成为一个流行的有机体，在不同的领域，由于其简单的生命周期，细胞生物学和遗传学的研究，缓解遗传分析，基因资源，原子核transformability和两个细胞器的增长和操纵。当遗传的方法是使用在交配菌株是一种常见的做法<em&gt; Chlamydomonass</em创建主导地位的营养分析二倍体，或四分体夹层，以确定核与细胞器的继承，测试等位，分析epistasy，或生成的图位克隆的目的的人群。此外，遗传杂交经常使用结合起来，特别是核基因型的细胞器基因型。在这里，我们展示了标准方法对配子，交配，合子萌发和四分体分离。该协议包括一个易于遵循的一系列步骤，这将使科学家所不熟悉的遗传方法适合<em&gt;衣</em&gt;。关键参数和故障点的解释。最后，提供进一步的信息和替代方法的资源。

Watch this Scientific Journal Video about Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis at JoVE.com

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

交配和四分体分离的遗传分析<em&gt;衣藻</em&gt;。在这里，我们展示了标准方法对配子，交配，合子萌发和四分体夹层。该协议包括一个易于遵循的一系列步骤，这将使科学家所不熟悉的遗传方法适合<em&gt;衣</em&gt;。

交配和四分体分离衣藻遗传分析

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

mating-tetrad-separation-chlamydomonas-reinhardtii-for-genetic

Research

JoVE Journal

Biology

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

19.9K 次观看.  Cornell University. 交配和四分体分离的遗传分析衣藻。在这里，我们展示了标准方法对配子，交配，合子萌发和四分体夹层。该协议包括一个易于遵循的一系列步骤，这将使科学家所不熟悉的遗传方法适合衣。

视频: Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

光/暗转换测试小鼠

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

科研

生物学

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

整装在鸡的抗体染色方法

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

在鸡胚神经诱导的含量为

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

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 ...