作者要感谢的合作与下面的科研资助机构的财政捐款:艾伯塔牲畜和肉类代理有限公司，猪肉CRC，艾伯塔猪肉，海波尔一个汉德克斯公司与NSERC CRSNG。

按照加拿大议会关于动物饲养的指导方针，并与学院动物政策及福利委员会批准 - 阿尔伯塔大学的畜牧，妊娠母猪由经过培训的人员大约一天收集妊娠和胚胎30安乐死。在过程中使用检查手套在所有时间。 1.样品采集和样品储存<ol><li>使用紧固螺栓接着放血安乐死母猪。戴上手套检查从每个安乐死收集母猪生殖道和胚胎。 注:样品和移植胚胎的快速收集到干冰或液氮，将导致更高质量的组织中其他分子的作品，如芯片和qPCR。 </li><li>解剖子宫，轻轻的胚胎盘胎膜内所有的胚胎从底层的子宫壁分离21。确保没有母体组织的污染。 </li><li>回覆冷冻前所有可行的胚胎线的长度和重量。 </li><li>收集每个人的胚胎，并立即与捕捉液氮冷冻前的铝箔包起来。 </li><li>转自液氮冷冻胚胎直接到-80℃的冰箱中。 </li></ol> 2.磨胚胎<ol><li>标记所有样品管（15ml）中与所需的样本数的样本ID加以分析。 </li><li>填充干冰热容器半满，并在干冰插入预标记的样品管中。 </li><li>戴上手套，冬天在上面另一对检查手套从-80转移预冷迫击炮和杵℃冰箱干冰的容器。 </li><li>放置在冷冻胚胎的干冰砂浆内。 </li><li>倾足够液氮覆盖胚胎和用杵成细粉研磨它。 </li><li>胚胎粉末转移到预标记的样品管与MICRospatula（图1），并放置在管中-80℃冷冻机中。 </li><li>使用干净预冷研钵每个胚胎和研磨每个胚胎，以避免样品间的交叉污染后改变考试手套。 </li></ol>采用改良氢氧化钠方法3基因组DNA制备方法<ol><li>标签所需的所有的样本数的离心管（1.5毫升），以进行分析。 </li><li>从之前的传递包含胚粉样品管-80℃冷冻到容器用干冰首发。 </li><li>吸管180微升的50mM的NaOH（氢氧化钠）到每个预标记的微量离心管中。 </li><li>打开培养箱和预热至95℃。 </li><li>用牙签胚胎粉末的正确量（约5-10毫克）（图2）从样品管转移到含有50mM的NaOH溶液的预标记的微量离心管中。 </li><li>拉üP上的同一牙签慢慢可视化所述DNA裂解物作为粘糊糊，白色透明状物质（图3）。 </li><li>用DNA裂解物转移离心管到预加热的培养箱中于95℃五分钟。紧随管转移到装满冰保丽龙箱。 </li><li>加入20μl的1M Tris-HCl中直接进入的离心管，并轻轻拍打该管混合。使用pH试纸，以确保pH在大约8.0（图4）之前，离心。 注意:当有大量样品制备的处理，是可以接受的随机挑选几样检查pH值。 </li><li>离心与DNA裂解液的管中，在2000 xg离心在微量两分钟，在室温下以除去未溶解的组织碎片。 </li><li>转移150微升顶部澄清上清液的进新管或96孔板中。 注:明确DNA裂解液准备在PC模板使用ř反应，这是在4℃下稳定两周，或者也可以保持在-20℃下一年。 </li></ol> 4.设计性别特异性PCR引物<ol><li>获得NCBI网站http://www.ncbi.nlm.nih.gov猪的性别决定区Y（SRY）登录号（NM_214452.3）和锌指蛋白X连锁（ZFX）基因（XM_005673501.1） /。 </li><li>复制这些NCBI登录号粘贴到一个在线的引物设计工具。 注意:最好的引物信息，包括长度，Tm和GC％以及PCR产物大小（bp）的将在计算机屏幕上产生的（图5）。 </li><li>验证的使用核苷酸BLAST程序（BLASTN）逆水猪基因组数据库104，以确保这些序列仅位于X和Y染色体为SRY和ZFX分别（图6）这些引物的特异性。 </li></ol> 5.基因组DNA PCR直接从DNA裂解液<ol><li>使用仅1微升的DNA裂解物作为15微升的PCR反应的模板的。 注意:可以在用多道移液器类似的方式来执行对96孔板的PCR的高通量筛选方法。 </li><li>请执行以下操作只是第一次PCR:通过添加购得已知性别的猪的基因组DNA 1微升（0.5纳克）准备一个PCR管无模板阴性对照和阳性对照两个附加管。以后，包括来自上一次成功的性别鉴定分析作为阳性对照的样品，并与用于性别确定的新PCR反应一起运行。 </li><li>使用任何热启动预拌的PCR酶，并通过添加引物制备主混合物并根据PCR反应的总数无核酸酶的水。加入1微升的DNA裂解成现有预PCR管与PCR主混合物的14微升。添加的引物，使得由两个性别特异性基因的引物的终浓度是在共有15微升的PCR为r的0.3微米反应的影响。 </li><li>设置以下PCR程序在热循环仪:95℃，3分钟，35个循环，每个为20秒熔化步骤98℃，接着在65℃15秒的退火步骤，接着是15秒延伸步骤在72℃。在最后的步骤，孵育1分钟的反应在72℃下，继续在4℃下孵育直至除去PCR管的用于凝胶电泳验证，以确定的性别。 注意:PCR条件和优化，根据在材料表中提到的PCR试 ​​剂盒的说明书。 </li><li>制备2％TBE琼脂糖凝胶时添加无毒绿光荧光SYBR DNA凝胶染色的适当的量。 注意:溴化乙锭可以代替绿光荧光染色，如果它是不可用的。 </li><li>添加1.5微升样染料（10倍）到PCR管中，并通过移液在PCR反应缓冲液向上和向下拌匀。 </li><li>负载10微升样品放入井并且r联合国琼脂糖凝胶具有适当的电压设置（在100伏的小胶设备，在150V 96孔凝胶装置上，直到染料带贯穿凝胶一半）。 </li><li>观察和利用激光扫描仪下的荧光设置调整频带的强度，并捕获凝胶的图像。注意:普通的凝胶成像仪可以取代的溴化乙锭的凝胶。 </li><li>通过识别与一个频带作为女性和两个频带作为男性（图7）的胚胎确定猪胚胎的性别。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

最相关的猪胚胎的DNA性别打字现有协议的仅适合于早期预注入阶段15,16。我们已经成功开发出在妊娠后期适用于猪胚胎筛选的协议。基于与从先前的研究6,18胚胎相似发育阶段的研究中，本协议被认为是安全的，成本低。 该协议也适于用于使用PCR通过DNA裂解物转移入96孔板性打字大量样品。板格式允许高通量筛选通过允许转印1μl的DNA的裂解物用于使用多道移液?...

国内生猪已成为发展，遗传学与营养在人类和畜牧部门的基本研究课题。猪作为生物体模型为人类研究的潜在可以归因于他们的生理相似之处。在家畜，性别比例操纵，提高选择和遗传改良计划1的有效性。性别鉴定单个胚胎是许多实验研究，包括但早期胚胎发育过程中2不限于基因，表观遗传学和性别二态性的X染色体失活使用的基本工具。 在小鼠的研究表明，产妇的饮食和其他因素可能导致男女比例失调3。在猪，性别比例失衡的原因包括父系品种4，子宫容量5，母猪的代谢状况6。因为在胚胎和窝中观察到的差异可以通过本身的影响xual异形，研究人员应借鉴有关他们的研究结论之前，了解胎儿的性别和性别比率。高效的工具和协议，在发展30日性别鉴定胚胎养猪的发展，将在这里讨论。 性分型的各种方法已经被开发用于在模式生物和家畜遗传研究。特别是在家畜，识别雌雄早期胚胎是一个非常普遍的做法，以提高遗传选择用于育种计划。早期胚胎用姬姆萨7或强烈的荧光8,9技术猪核型已被用于性别特征。然而，这些方法是耗时，不适合快速，准确地筛选大量的胚胎。 最有效的性别分型方法是使用热稳定DNA聚合酶和一对引物DNA扩增。通过PCR方法DNA性别鉴定是比较具体的，快速，灵敏，邻NLY需要接入蜂窝材料的微量。是对人类进行的10首基于PCR的胚胎性别鉴定，后来在小鼠11，12牛，水牛13和 14只羊植入前胚胎。在猪，通过单一对Y染色体特异性DNA引物15，建立了植入前胚胎早期的DNA性别分型方法。然而，从雄性特异性SRY基因16和位于X和Y染色体17上的锌指基因的非性判别区域的Y染色体选择用于性别确定的最常见的PCR引物。随后，这些引物已经被应用，以确定在这项研究中具有改进的引物特异性日30的胚胎的性别来检测仅一个锌指基因的X染色体。 从猪植入前胚胎基因组DNA可以通过将一个完整的胚泡与proteinas缓冲提取Ëķ16或通过采取一些细胞活检从个体早期卵裂胚胎15并将它们用于直接PCR。然而，从猪血液，毛发，组织或大小超过几厘米大孕的DNA释放不能有效地使用蛋白酶K法进行。使用任一耗时苯酚/氯仿协议6或昂贵基于列的试剂盒18已经建立了这些材料的DNA提取方法。为了避免使用可能有毒的化学品，有开发价格低廉，容易和自由酚DNA提取方法的趋势。这对PCR的质量的基因组DNA，从鼠标 19和斑马鱼20组织中的隔离型协议的已使用热氢氧化钠和三（能手）成立。这项研究提供了一个协议来获取DNA与PCR性爱能手修改和重新设计的复式引物对直接从日细胞裂解液30猪胚胎打字精度高。

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			<td height="26" style="height:27px;width:331px;">KAPA HiFi HotStart Ready Mix PCR kit&#160;</td>
			<td style="width:203px;">KAPABiosystems</td>
			<td style="width:189px;">KR0370</td>
			<td style="width:463px;">other Hot Start Taq polymerase can be used after optimization</td>
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			<td height="26" style="height:27px;width:331px;">SYBR Safe DNA Gel Stain</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:189px;">S33102</td>
			<td>Ethidium bromide can be substituted for SYBR Safe</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Pig female and male genomic DNA</td>
			<td style="width:203px;">Zyagen</td>
			<td style="width:189px;">GP-160-F1 &#38; GP-160-M5</td>
			<td>Postive controls from the tissues of known sex DNA can be used.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Typhoon FLA 9500 laser scanner</td>
			<td style="width:203px;">GE Healthcare Life Sciences</td>
			<td style="width:189px;">28-9969-43</td>
			<td>other imaging system can be used</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Free Soft Nitrile Examination Gloves</td>
			<td style="width:203px;">WWR</td>
			<td>89038-270</td>
			<td>any other examination glove can be used</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Sodium hydroxide, solid&#160;</td>
			<td style="width:203px;">Fisher</td>
			<td style="width:189px;">BP 359 -212</td>
			<td>Molecular Biology Grade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Eppendorff DNA LoBind Tubes, 1.5 ml, PCR clean</td>
			<td style="width:203px;">Eppendorff</td>
			<td style="width:189px;">0030 108.051</td>
			<td>heat resistant</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">ThermoStat plus</td>
			<td style="width:203px;">Eppendorff</td>
			<td style="width:189px;">22670204</td>
			<td>Use as a incubator for 95C, don&#39;t need to use the heater</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Toothpick</td>
			<td>Bunzl Plc</td>
			<td style="width:189px;">75200815</td>
			<td style="width:463px;">Any round wooden toothpicks can be used - quality wood</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Microcentrifuge 5417R/5417C</td>
			<td style="width:203px;">Eppendorff</td>
			<td style="width:189px;">22621807</td>
			<td>This model was discontinued. But another newer model can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Microspatula</td>
			<td style="width:203px;">Fisher</td>
			<td style="width:189px;">SDI28540115</td>
			<td>Autoclaved before use each time.</td>
		</tr>
	</tbody>
</table>

accurate and phenol free dna sexing of day 30 porcine embryos by pcr

研究中的猪产前编程已经表明，发育中的胚胎或胎儿的性别可以影响发育结果。因此，确定胚胎的性别的能力是必要的，许多实验特别是关于早期发展。本协议表明猪基因组DNA用PCR使用一种廉价的，快速的和无毒​​的制剂。第30天的胚胎必须按照机构动物政策及福利委员会在本协议确定的方针得到人道收集。整个胚胎此基于PCR性别鉴定技术制备简单地涉及使用预冷研钵和杵研磨冷冻的胚胎成细粉。 PCR的质量DNA从胚胎粉末少量通过在碱性裂解试剂施加热孵育释放。接着，将DNA溶液与中和缓冲液混合，并直接用于PCR。两个引物对产生的DETEC吨特定性别确定Y-染色体（SRY）和在X染色体以高精度和特异性的ZFX区域的区域。相同的协议可以被应用到其它细长胚胎（第10天至14天）比日早30.此外，此协议可与96涌出板筛选大量的胚胎时，使之成为自动化和高可行进行吞吐量性别特征。

从345的DNA裂解物通过PCR筛选性别决定的代表性结果示于图7和表 1中总结。 如在图7中可以看出 ，引物退火温度在65℃是在不同样品之间的这种PCR方案产生类似的强度和谓词的扩增子的大小（图5）的最佳条件。 SRY（400?...

Watch this Scientific Journal Video about Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR at JoVE.com

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

30日猪胚胎的准确和无酚DNA性别鉴定的PCR

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, ...

accurate-and-phenol-free-dna-sexing-of-day-30-porcine-embryos-by-pcr

Research

JoVE Journal

Developmental Biology

9.9K 次观看.  University of Alberta. This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

视频: Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

染色质监管机构和各国的全基因组快照<em&gt;爪</em&gt;通过胚胎的ChIP-SEQ

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

科研

发育生物学

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.

A method for fluorescent staining of fixed biological material with the specific DNA label methyl green is described. Methyl green is used in a diluted aqueous solution and is very resistant to photobleaching. Its far-red emission allows for deep specimen imaging, making it particularly adequate for whole embryos.

DNA的特定染色甲基绿在胚胎的荧光标记的应用

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

方法细胞或DNA的精确本地化转移到着床后早期小鼠胚胎

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

联体斑马鱼胚胎一代发展囊胚的手术融合

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

使用层粘连蛋白521矩阵整合人诱导多能干细胞的自由衍生

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

用高频超声 (30/45 mhz) 系统对胎儿小鼠进行心血管成像

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

斑马鱼胚胎和幼虫的整个山免疫性化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...