S. mGFP小鼠阿伯尔和批判地阅读文件，并提供技术援助的Zervas实验室的成员，我们非常感谢。答：布朗支持大脑科学卡普兰夏季研究生研究奖。 E.诺曼德是支持的脑科学计划的研究生研究奖。这项研究是由启动研究经费（MZ）。

他莫昔芬的制备及灌胃程序（E8.5） <ul><li>通过溶解在他莫昔芬预热玉米油，准备了他莫昔芬20毫克/毫升的原液。 </li><li>解决方案在37℃，2小时一个nutator和涡间歇。 </li><li>他莫昔芬的股票免受光，储存在4 ° C，并使用长达1个月。 </li><li>命运映射实验，建立一个养殖对瑞士韦伯斯特女性（野生型;从Taconic公司购买）组成的和男性携带一个基因的特定CreER T等位基因和记者等位基因。出于演示的目的，我们使用Wnt1 - CreER 吨 ; mGFP男性（Ellisor 2009年）。 </li><li>每天早晨检查瑞士韦伯斯特女性阴道塞的外观。候为0.5天，性交后阴道塞的一天上午（0900）和日期计算他莫昔芬管理基于这个出发点。 （在这个实验中，使用胚胎每天8.5）。 </li><li>使用动物饲养针（20克x 1-1/2）1毫升注射器制订的他莫昔芬原液（4毫克他莫昔芬）200μL。 </li><li>坚决抑制抓颈背的时候怀孕的瑞士韦伯斯特女性和固定头部和翻身所以腹侧的一面朝上。 </li><li>保持自由手指之间的尾巴保持身体在一条直线上。 </li><li>放置到嘴角的喂养针，轻轻地引导屋顶沿口针。 </li><li>旋转针头，这是对身体平行，而同时倾斜的头部，背部保持颈部挺直。 </li><li>指南上下针，食管向胃。要小心，不要进入气管。如果有阻力针，动物的咽反射从事，或如果鼠标斗争，立即取出针，并再次尝试灌胃。 </li><li>一旦喂养针在肚子里，管理进肚子里的他莫昔芬和解剖之日起，直到返回到她家笼女。 </li></ul>开颅手术： <ul><li> Intracardially灌注成人的命运映射与4％的煤灰鼠标，并用剪刀除去头只以上的肩膀脊柱切割。 </li><li>沿背中线头（喙尾鳍）穿过头皮和揭露的scull的运行手术刀。 </li><li>使用手术刀，刮走沿着头骨侧面和后部的任何多余的组织或肌肉。 </li><li>镊子，穿刺只是延髓的嗅球，并创建了一个小洞，以容纳细剪刀提示中线的头骨。 </li><li>插入此孔的罚款剪刀和一个切口内侧向外侧约嗅球长度。这一削减将打破在鼻骨和额骨的交集的头骨，并提供良好的剪刀的访问。 </li><li>沿矢状一定要保持远离大脑的角度，以避免损坏的根本组织剪刀提示（背中线）在颅骨缝线切割。 </li><li>轻轻抓住沿内侧切口颅骨剥离钳和骨暴露大脑。头骨可能芯片小块或打破在较大的部分。继续使用镊子，以消除所有的额叶，顶叶，interparietal，和枕骨骨。 </li><li>破解套paraflocculi（沿水平小脑位于大脑外侧边缘），每边捏的球状骨的骨头。轻轻地拉开了距离的骨头。 </li><li>转动头部倒挂（背面朝下），并使用镊子断绝颅神经和大脑释放的头骨。 </li></ul>全山显微镜：成人的大脑<ul><li>评估成人大脑的GFP标记用荧光解剖范围。 </li><li>脑转移到培养皿中含有PBS和照片使用PictureFrame或其他适当的软件。 </li></ul>整个山显微镜：E12.5胚胎<ul><li>评估的GFP标记的整装胚胎，用荧光解剖范围。 </li><li>转让那些GFP整个安装到培养皿中含有PBS和照片使用PictureFrame或其他适当的软件积极。 </li><li>使用镊子夹断的尾巴从每个胚胎中的一小片，放置在每一个PCR管片和基因型的组织使用两个等位基因PCR（Ellisor 2009），以确认通过整装分析中看到的结果。 </li></ul>显微解剖和E12.5胚胎腹侧中脑外植体的制备<ul><li>继剥离子宫链和ID整个安装荧光，转让冰冷的无菌PBS在细胞培养皿中的胚胎的命运映射胚胎entification。 </li><li>用细剪刀，取出的胚胎切割横头部分，尾椎菱1。 </li><li>接下来，删除，透过间脑冠状切开​​的喙头部分。这将会使一个筒状结构，其中第四脑室通过脑神经囊泡形成一个背，腹组织之间的渠道。 </li><li>小心地插入剪刀的提示，进入第四脑室，并剪断，沿背中线，尾椎到延髓，全面开放管，创造一个“开卷”的准备。现在暴露腹侧中脑内侧，而将驻留的中脑背侧的两个半横向。 </li><li>下面的腹侧中脑组织打下更断然删除任何剩余的非神经组织。如果需要，请在延髓和尾鳍等方面以及背的“翅膀”的外植体平放的凹槽。外植体，现在应该像“蝴蝶，其中背侧中脑代表的翅膀，和菱1尾部。 </li><li>为了进一步孤立的流式细胞仪分析细胞培养实验的腹侧中脑，删除的横向组织（背方面）。 </li></ul>代表性的成果： 在这个GIFM实验中，Wnt1表达细胞是永久和heritably标记胚胎发育过程中，他莫昔芬在E8.5管理Wnt1 - CreERT; mGFP胚胎。随后，这些标记的细胞，是通过整装的荧光和部分免疫组化法 ，以确定如何Wnt1衍生细胞对神经结构的跨越发展可视化。整装荧光依赖于膜结合GFP的mGFP记者组成部分，因此，提供细胞的信息，以及Wnt1源性神经预测的总体看法。例如，与他莫昔芬在E8.5，Wnt1 CreERTmGFP在E12.5展览，主要的中脑，后后脑和脊髓（图1A）绿色荧光的胚胎管理。在高放大倍率，精细的神经元预测支配的脑，体壁，四肢和颅面地区。在这一发展阶段，胚胎是半透明的，内部的GFP标签是很容易看出端倪。然而，由于大脑的发育，成熟的组织密度掩盖内部的大脑结构所产生的绿色荧光。因此，与他莫昔芬在E8.5管理，只有轻微的绿色荧光蛋白标签是在优越的整装荧光（图1C，插图）成人丘观察。此外，一些神经轴突的预测是沿后脑腹面窃喜（未显示）。相反，当他莫昔芬是在E9.5管理，出现大量的绿色荧光蛋白标签整个伪劣丘（图1B，插图）。在整个装载荧光的增加可能表明 Wnt1表达细胞在E9.5作出更大的贡献明显背脑浅表地区或延长这个区域内，而不是从E8.5细胞Wnt1表达更多的进程。无论如何，这在整个装载荧光Wnt1表达的差异，反映在开发过程中的动态性质，并演示了如何GIFM可以被用来追踪从胚胎到成年不同的细胞群。 随着免疫组织化学，从Wnt1 CreERT的命运映射的组织切片 ; mGFP动物在细胞水平上进行了分析。 GFP和β-半乳糖苷酶（β-半乳糖苷酶）抗体结合各种组织特异性的生物标志物来确定罚款规模Wnt1谱系的细胞神经系统结构和细胞类型的贡献。他莫昔芬管理E8.5和E12.5组织分析，双阳性细胞（绿色荧光蛋白和β-半乳糖苷酶）是观察整个窃喜预测通过腹侧中脑（图1B）中脑和后脑。 Wnt1宗族标记在E8.5在成人大脑，产生了许多脑结构的优势和劣势丘，以及在附近的腹侧被盖区和黑质（图1D）的多巴胺能神经细胞群（见也Zervas 2004年）。相比之下，在E9.5标记的Wnt1系引起下丘，以及在多巴胺的细胞群（图1F）附近的神经元。 <img src="/files/ftp_upload/1687/1687fig1.jpg" alt="图1" /> 图1 Wnt1的命运映射代表性的结果。 整个安装和免疫细胞化学结果Wnt1 - CreERT的例子; mGFP胚胎和成人大脑的命运E8.5（AD）的映射（标记）E9.5（EF）。答：Wnt信号1 CreER; mGFP胚胎E12.5 GFP荧光的展览，主要在中脑（MB），后后脑（HB）和脊髓（SP ）。 B.标记的细胞，可视化和分析免疫组织化学在细胞水平上，在这1微米厚的光学部分（40X目标，Z系列收购）所示。核β-半乳糖苷酶（nβ- GAL）抗体标记（红色）和GFP抗体标记（绿色）表示命运映射显示在腹脑细胞。在这里，重组细胞双阳性（nβ-半乳糖苷酶+ / GFP +）因为条件 mGFP记者的性质。 C.全装载荧光显微镜，揭示了成人优越丘（SC）（插图）淡淡的绿色荧光。评估Wnt1血统上标注的成人节E8.5低倍率（5倍）显微镜表明，Wnt1血统引起脑的结构，包括上级（SC）和劣势（IC）丘（C ） D.一个40X，1毫米的光学部分在腹侧中脑多巴胺能神经元nβ-半乳糖苷酶+细胞和丰富的GFP +轴突丛附近。 E.整装荧光（插图）的脑下丘，很容易观察到的大量的绿色荧光蛋白标签E9.5结果标记。 Wnt1血统上标注的成人节（β-半乳糖苷酶+，红）在E9.5集中在低倍率（5倍）显微镜（五）下丘（背后路脑）。 F.一个40X，1毫米的光学部分在腹侧中脑多巴胺能神经元nβ-半乳糖苷酶+细胞和丰富的GFP + 轴突丛附近。 E8.5与E9.5标记Wnt1源性神经元正逐步从背脑限制，而Wnt1血统坚持促进腹脑。

<ol>
	<li>Ellisor, D., Koveal, D., Hagan, N., Brown, A., Zervas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+conditional+reporter+alleles+in+the+developing+embryo+and+embryonic+nervous+system.">Comparative analysis of conditional reporter alleles in the developing embryo and embryonic nervous system.</a> Gene Expr Patterns. 9, 475-489 (2009).</li><li>Zervas, M., Millet, S., Ahn, S., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+behaviors+and+genetic+lineages+of+the+mesencephalon+and+rhombomere+1.">Cell behaviors and genetic lineages of the mesencephalon and rhombomere 1.</a> Neuron. 43, 345-357 (2004).</li></ol>

所有作者同样这项工作作出了贡献，按字母顺序列出。每一个人的贡献是明显的他或她参加在视频文章。

GIFM系统，我们已经证明可以用于广泛的多种细胞过程中的问题的答案。例如，具有不同的Cre司机工程小鼠可以用来针对任何类型的细胞，组织或利益的遗传谱系。此外，因为他莫昔芬可在任何时间点胚胎或产后管理，重组，可以针对任何相关的发展阶段。该系统的空间和时间的控制是研究如何表达特定基因的细胞结构或地区的利益，以及细胞迁移和图案在开发过程中的事件作出贡献的理想。 ...

a practical approach to genetic inducible fate mapping: a visual guide to mark and track cells in vivo

命运的地图生成标记和跟踪体内细胞，以确定如何祖贡献的具体结构和细胞类型，在发展中国家和成人组织。在这个概念的发展是 G enetic我nducible F 吃中号apping（GIFM），将基因表达，细胞的命运，并在体内的细胞行为，创建基于遗传谱系的命运地图。 GIFM战功的的X CreER线，X是一个基因或基因调控元件的设置，赋予一个修改后的噬菌体蛋白质，Cre 重组酶（CreER T） 的表达空间 。CreER T包含一个修改的E strogen ř eceptor配体结合域呈现CreER T扣押在细胞质中的药物他莫昔芬的情况下。他莫昔芬发行CreER T，即转移到细胞核与调解由loxP位点两侧的DNA序列之间的重组结合。在GIFM，重组之间，通常会出现一个loxP位两侧停止前，如GFP报告基因的卡带。 小鼠繁殖包含一个区域或特定细胞类型的 CreER和有条件的记者等位基因。未经处理的小鼠不会有标记，因为在记者停止卡带防止进一步的报告基因的转录。我们管理灌胃定时怀孕的女性，这提供CreER T版本和随后易位到细胞核去除记者从停止磁带的时间控制的他莫昔芬。重组之后，记者等位基因的组成和heritably表达。这一系列事件标志着细胞等，他们的遗传史是不可磨灭的记录。重组记者，作为一个高保真的遗传谱系追踪器，一旦上，从最初用来驱动CreER T基因表达耦合。 我们申请GIFM在小鼠成年细胞类型和组织的遗传谱系研究的正常发展和确定贡献。我们也使用GIFM遵循细胞突变的遗传背景，以更好地了解复杂的表型，模仿人类遗传性疾病的显着特点。 此视频文章指南，通过实验方法的研究人员成功申请GIFM。我们证明的方法，使用特点Wnt1 CreER T;由 mGFP小鼠胚胎一天（五）8.5管理通过清扫在E12.5和epifluorescence立体显微镜分析灌胃他莫昔芬。我们还演示了如何微为外植体的制备或流式细胞仪分析解剖的命运映射域和成人命运映射的大脑解剖整装荧光成像。总的来说，这些程序允许研究人员在发育生物学和疾病模型解决的关键问题。

Watch this Scientific Journal Video about A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo at JoVE.com

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

遗传诱导的命运映射（GIFM）标记和细胞与精致的空间和时间的控制轨道<em&gt;在体内</em&gt;和阐发了如何从一个特定的遗传谱系细胞有助于发展中国家和成人组织。这里展示的是命运之图E12.5小鼠胚胎啶和外植体分析所需的技术。

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-practical-approach-to-genetic-inducible-fate-mapping-visual-guide

Research

JoVE Journal

Biology

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

16.7K 次观看.  Brown University. 遗传诱导的命运映射（GIFM）标记和细胞与精致的空间和时间的控制轨道在体内和阐发了如何从一个特定的遗传谱系细胞有助于发展中国家和成人组织。这里展示的是命运之图E12.5小鼠胚胎啶和外植体分析所需的技术。

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

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 ...

科研

生物学

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. ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

一个反向遗传学方法测试胚胎发育过程中的功能冗余

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

单细胞在斑马鱼的命运映射

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

实用指南系统发育的非专家

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

药物与DNA相互作用的细胞全基因组映射与COSMIC（小分子交联，染色质隔离）

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...