我们非常感谢南希Papalopulu 爪电击的援助和柏彦BONEV 。我们也感谢马克 - 迪翁的关键读，杰里米绿色有益的讨论和刘实验室成员的支持和约翰沃林福德。这项工作是由BBSRC（BB/E013872/1）和威康信托基金会（081880/Z/06/Z）KJL补助。

1A部分。设备 显微镜：直立的立体解剖与低功耗的目标范围<ul><li> 电压/脉冲发生器：BTX ECM 830方波电穿孔系统</li><li> 移液器拉马：P - 87微管拖轮（萨特仪器公司，CA） </li><li> 机械手：筹备工作粗糙，或合并的粗，细。 </li><li> 微管持有人：精细的科学工具</li><li> 电极：自制</li><li>自制电穿孔室： </li></ul> “L”形的电极： <ol><li>切8厘米高纯度的0.4毫米的钨丝（古德费洛）和中点处贴上一个1ml注射器，使用腻子（我们使用的Blu - TACK）。离开4厘米钨丝，从暴露成“L”形的注射器和弯尖的一角，从一端1厘米（图1A）。 </li><li>修剪提示，以便最终措施，长0.5毫米。 T他的提示是电极总站。 </li><li>过剩的钨丝平行运行注射器，并用它来连接电极的脉冲发生器。 </li><li>重复过程中，一对电极。 </li><li>方波脉冲发生器通过直流电缆连接电极。 </li></ol>电穿孔室<ol><li>线的底部为90毫米〜5毫米的非有毒橡皮菜。 </li><li>填写电击媒体的菜。 </li><li>使用5号制表钳刻出一个T形好（图2）。长以及应测量1〜2毫米x 2毫米× 10毫米，短1〜2毫米x 2毫米x 5毫米。可水洗的电盘和重用。 </li></ol>第1B部分。试剂 DNA或收取大分子<ul><li> 微量移液器：1毫米宽4“长高硼硅玻璃毛细管（WPI的，TW100 - F） </li><li> 文化传媒：正常两栖类媒体（不结盟运动） </li><li> &gt; 10倍的股票：1100毫米氯化钠，20毫米KCL，10毫米的Ca（NO 3）2•4H 2 O，1 mM EDTA的。 <ul><li>在4 ° C的高压灭菌器和存储</li><li> 1 + X南：碳酸氢钠3 0.1毫米和0.2毫米娜3 PO 4的稀释10倍的股票，缓冲区。 </li></ul></li><li> 非洲爪蟾蝌蚪阶段28 </li></ul> DNA的制备： <ol><li>准备表达质粒，使用标准协议。 </li><li>重悬DNA的一个终浓度为1μg/μL核酸自由H 2 0。 </li></ol> *我们有包含一个强大的CMV启动子的载体，如pCS2，成功+ [16]。谱系分析，我们通常包括在终浓度为0.1微克/μL的DNA编码绿色荧光蛋白（pCS2 GFP）的。 [0.1-3微克/μL的DNA浓度进行了测试。我们发现，浓度低于0.8μg/μL的低效标记的细胞，而DNA浓度大于2微克/＆MU;升不提高电穿孔效率]。 吗啉寡聚核苷酸的准备： （注：。MOS需要fluoresceinated（3&#39; -羧基改性）或以其他方式收取） <ol><li>悬浮吗啉代寡核苷酸（MOS）（Genetools， <a href="http://www.genetools.com" target="_blank">www.genetools.com</a> ）核酸自由H 2 0浓度为2毫米。 </li><li>热原液分装在65 ° C 5分钟。 </li><li>稀释至终浓度为0.5毫米核酸自由水。 </li></ol> * 0.1 - 1mm的MO解决方案进行了测试。 0.5毫米MO解决方案足够的许多间质细胞的电穿孔。 微量移液器<ol><li>准备从高硼硅玻璃毛细管（宽1毫米，长4“，WPI的TW100 - F）。微量使用针拉马准备8-12毫米长的锥度和精尖的微量。 </li><li>粉碎提示〜2毫米从尖镊子，创造一个锯齿状的突破。 </li></ol>媒体<ol><li>孵化媒体：准备新鲜的3 / 4正常两栖类媒体（NAM）从1x股票。添加0.025毫克/毫升的庆大霉素。 </li><li>电穿孔媒体：如上所述，用0.1％苯佐卡因（Sigma公司，06950）。 </li></ol> 2。电穿孔微管安装<ol><li>填写微量〜1μL注射液。 </li><li>固定在显微操作的微管和微量（Picospritzer二）重视。 </li><li>从桌面上，在50 °的角度微量。 </li><li>设置注射压力20 PSI。 </li><li>校准微量注入脉冲NL 30％。 </li></ol>蝌蚪准备<ol><li>麻醉阶段28 爪蟾幼虫孵化5分钟电击媒体。 </li><li>转移到充满电穿孔媒体电击室麻醉蝌蚪。位置胚胎内长好，让头部背侧和腹侧外露的丁字路口的责任。尾巴相比，头部应该稍微升高。 </li><li>使用镊子，轻轻安全以及与周围橡皮蝌蚪。 （注：如果是没有担保的蝌蚪，它可能会抽搐和联系，在电穿孔电极，在这种情况下丢弃的蝌蚪面巾纸将严重受损。） </li></ol>电穿孔<ol><li>插入微量提示后水泥腺和面部间质成。 </li><li> 30 NL溶液注入到间质。 </li><li>缩回微量。 </li><li>快速对齐平行电极提示胚胎的头（图3）。 </li><li>适用于8月50毫秒，20mV的方波。 </li><li>缩回电极。 </li><li>使用镊子小心释放蝌蚪转移到3 / 4的不结盟运动，0.025毫克/毫升的庆大霉素。 </li><li>蝌蚪可以在不结盟运动，0.025毫克/毫升overn的3 / 4孵育的飞行，或更长的时间。 </li><li> 24小时后，荧光显微镜高效电击屏幕胚胎。 </li></ol> 3。代表性的成果： 允许使用荧光分子电穿孔胚胎容易甄别。图4显示了一个典型的一批MO电穿孔蝌蚪〜12，48和96小时后，电穿孔，孵育在14.5 ° C。用荧光显微镜，MOS可电击后立即可视化，坚持几天后电击。根据我们的经验，荧光弱明显的阶段，46（〜5天以后）。在软骨，荧光分化（42日）后开始急剧下降，但是，莫荧光仍然存在较为强烈的其他类型的细胞，如咽部内胚层。荧光显微镜显示，纳入到几个颅面组织，包括软骨寡核苷酸。寡核苷酸荧光彗星一个经常在组织上的头两侧可视化。这可能是由于整个宽松的颅面间质注射液，以电穿孔前迅速扩散。 <img alt="图1" src="/files/ftp_upload/3381/3381fig1.jpg" /> 图1自制的电极。 L形的钨丝，连接到一个1 ml注射器，采用无毒的粘土或腻子。 （一）电极总站措施5毫米。 （二）将一对电极，这样的总站并行运行。电极连接到脉冲发生器，直流电缆。 <img alt="图2" src="/files/ftp_upload/3381/3381fig2.jpg" /> 图2电穿孔室。 （一）90毫米菜用橡皮衬里填充与媒体和T形腔刻有没有5钟表匠的镊子。 （二）长期方面的措施2毫米x 2毫米X10毫米而2毫米x 2毫米x 5毫米的短期措施。胚胎的头枕在丁字路口，腹侧。 <img alt="图3" src="/files/ftp_upload/3381/3381fig3.jpg" /> 图3示意图说明电击过程。圣28蝌蚪是摆在电击室，腹一面向上。微管插入面部间质底层水泥腺。注入。微管被删除，L形电极平行侧翼头。应用8个50毫秒，20 mV的方波。缩回电极。蝌蚪生长所需的阶段。 Visualise MOS或GFP的表达，用荧光显微镜。 <img alt="图4" src="/files/ftp_upload/3381/3381fig4.jpg" /> 图4蝌蚪代表12（A），48（二），96（三）小时后电击（阶段30，分别为34和44） 。 （A“B”），可以可视化荧光莫内颅面间质在雄鹿ES 30和34。荧光可以检测阶段44（箭头，C&#39; - C“）的软骨。肠道高度autofluorescent。

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作者有没有利益冲突。

在这段视频中，我们已经证明爪蟾蝌蚪的面部间质电穿孔介导的基因传递的可行性。使用这种方法，我们可以绕过早期发育的影响，操纵基因的功能，让我们在以后的时间点目标特定的组织。我们的研究表明，可能会影响颅面间质细胞的异质人群，让我们研究的电穿孔细胞以及宗族利益的蛋白质细胞的自治要求。结合实时成像，我们可以使用这种方法来研究基因功能，随着时间的推移，在颅面发育。这种新颖的方法，突出器官的研究非洲爪蟾的可追踪性。我们预计，这种方法可广泛适应研究以及其他组织的形态发生和分化。

A correction was made to <a href="http://http://www.jove.com/video/3381">Electroporation of Craniofacial Mesenchyme</a>. There was an incorrect unit used for the voltage.

Erratum: Electroporation of Craniofacial Mesenchyme

electroporation of craniofacial mesenchyme

电穿孔是一种有效的方法，在精确的时间点的组织和确切地点提供DNA和其他带电的大分子。例如，电已经取得了巨大成功用于研究在爪蟾神经和视网膜发育，鸡和鼠标1-10。然而，重要的是要注意，在所有这些研究中，研究者针对软组织。因为我们是在颅面发育有兴趣，我们适应目标面部间质的方法。 当我们进行文献检索，我们发现，给我们带来惊喜，很少有成功的基因转移到软骨组织的报告。这些研究大部分基因治疗研究，如siRNA或蛋白质传递到软骨细胞株，或关节炎的动物模型 11-13 。在其他系统，如鸡或鼠标，电击面部间质已具有挑战性（个人COMMUNICAT离子，颅面发育，KCL部）。我们推测，到爪蟾 procartilaginous和软骨组织的电击可能更好地工作。 ，我们在我们的研究表明，有效的基因转移到面部软骨发生在早期阶段（28），面部原基仍是软组织组成，前软骨分化。 爪蟾是一个十分便利的分析颅面部发育的脊椎动物系统。颅面结构可见，在爪蟾更容易比其他任何脊椎动物模式，主要是因为非洲爪蟾胚胎受精的外部，允许的最早阶段的分析，并促进在单细胞分辨率的实时成像， 以及重用的母亲14。 爪蟾在外部发展的脊椎动物模型，是更多有用的颅面分析比斑马鱼，爪蟾幼虫都较大，更容易DIssect，和面部区域的发展是更容易获得比在鱼相当于地区成像。此外， 非洲爪蟾是对人类进化上更接近比斑马鱼（〜100万年前接近）15。最后，在这些阶段， 爪蟾蝌蚪是透明的，并发表达荧光蛋白或分子将允许开发软骨容易可视化。我们预计，这种做法将让我们能够迅速，高效地测试候选分子在体内模型系统。

Watch this Scientific Journal Video about Electroporation of Craniofacial Mesenchyme at JoVE.com

Electroporation of Craniofacial Mesenchyme

颅面软骨发展与其他组织的密切联系和难以操作，在活的动物。我们使用的是电击期间提供的颅面骨骼生长的分子工具，而绕过早期胚胎的影响。这种方法将使我们能够有效地测试候选分子<em&gt;在体内。</em

颅面间质细胞的电

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

electroporation-of-craniofacial-mesenchyme

Research

JoVE Journal

Biology

11.8K 次观看.  King's College London. 颅面软骨发展与其他组织的密切联系和难以操作，在活的动物。我们使用的是电击期间提供的颅面骨骼生长的分子工具，而绕过早期胚胎的影响。这种方法将使我们能够有效地测试候选分子在体内。

视频: Electroporation of Craniofacial Mesenchyme

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

电穿孔分枝杆菌

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

科研

生物学

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

评估外胚层上皮细胞在颅面发育的信号属性

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

在OVO在鸡胚视网膜电穿孔

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

分离的小鼠胚胎面外胚层和间充质

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

颅面部发育中的SOX10可视化：使用枫转基因斑马鱼线时间推移共聚焦显微镜

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

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 ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

评估具体物种的贡献颅面开发利用鹌鹑，鸭嵌合体

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...