Name: manipulation of gene function in mexican cavefish
Uploaded: 2019-04-22T13:00:00
Description: Watch this Scientific Journal Video about Manipulation of Gene Function in Mexican Cavefish at JoVE.com

作者感谢 Sunishka Thakur 在基因分型和成像图2中描述的考2 突变鱼方面提供的帮助。这项工作得到了国家科学基金会 (NSF) 对 a. c. k. 的1656574奖、国家科学基金会授予 j. k. 和 a. c. k. 的1754321奖, 以及国家卫生研究院 (NIH) 授予 R21NS105071 a. c. k. 和 e. r. d。

1. 莫菲洛里诺寡核苷酸设计
注: 通过国家生物技术信息中心 (NCBI) 基因和 NCBI SARA (https://www.ncbi.nlm.nih.gov) 以及 Ensembl 基因组浏览器 (https://www.ensembl.org) 提供了a. mexicanus的序列。在设计用于表面和洞穴居住形式的吗啡时, 在这一阶段识别形态之间的任何遗传变异是至关重要的, 因此可以避免这些遗传区域成为吗啡的目标。吗啡目标站点内的任何多态性变化都可能导致无效的?...

在这里, 我们提供了一种使用吗啡、CRISPR/Cas9 基因编辑和转基因方法来操纵基因功能的方法。斑马鱼的丰富基因技术和这些系统的优化, 很可能会使这些工具轻松地转移到中.最近的发现在mexicanus中使用了这些方法, 但在调查该系统中的各种形态、发育和行为特征时, 它们仍未得到充分利用。,</s...

自达尔文的物种起源1 以来, 科学家们已经深入了解了由于洞穴生物2的作用, 这些特性是如何进化形成的, 以应对确定的环境和生态压力.墨西哥的特拉, a. mexicanus, 包括眼睛祖先的 "表面" 人口居住在整个墨西哥和南部的得克萨斯州的河流, 以及至少29个地理上孤立的洞穴形态种群居住在塞拉德阿布拉和墨西哥东北部其他地区 3。一些与洞穴相关的特征已经被发现在一个维菌, 包括改变的耗氧量, 脱色, 眼睛的损失, 和改变喂养和觅食行为4,5, 6, 7,8,9。由于清晰的进化史、对生态环境的详细描述以及独立演化洞穴的存在, mexicanus为研究收敛进化的机制提供了一个强有力的模型人口10,11。在洞穴中存在的许多洞穴衍生特征, 包括眼睛丢失、睡眠不足、增加喂养、失去学业、减少攻击性和减少压力反应, 都是通过独立的起源多次进化的, 通常使用洞穴8,12, 13,14,15之间的不同遗传途径。这种反复的进化是一个强大的方面 , 可以提供洞察更普遍的问题 , 遗传系统可能会扰产生类似的表型。
虽然基因技术在许多鱼类 (包括鱼) 基因功能机械调查中的应用有限, 但斑马鱼的最新进展为鱼类的基因技术发展提供了基础16,17,18,19,20。许多工具被广泛用于斑马鱼操纵基因表达, 这些程序的实施早已标准化。例如, 在单细胞阶段注射吗啡寡核苷酸 (mo) 有选择地阻断 rna, 并在开发过程中阻止短时间窗口的翻译 21, 22.此外, 基因编辑方法, 如聚集定期间隔的短回文重复 (CRISPR)/crispr 相关蛋白 9 (Cas9) 和转录激活剂样效应核酸酶 (TALEN), 允许产生定义的缺失或在某些情况下, 通过基因组19、20、23、24 的重组进行插入。转基因是用来操纵稳定的基因表达或功能在细胞类型的特定方式。Tol2系统有效地用于生成转基因动物, 方法是将转座酶 mrna 与含有转基因 25,26的tol2 dna质粒进行共置。Tol2系统利用 Medaka 的tol2移位酶生成具有稳定性的转基因结构的种系插入。产生Tol2转基因包括为tol2转座酶17注入含有由Tol2整合位点和 mrna 两侧的转基因的质粒.该系统已被用来生成一系列的转基因线斑马鱼, 它的使用最近已扩展到其他的紧急模型系统, 包括鱼, 小鱼, 粘背, 以及最近, 墨西哥洞穴 27, 28,29,30。
虽然紫鱼是一个迷人的生物系统, 用于阐明性状进化的机制, 但它作为进化模型的全部能力尚未得到充分利用。这在一定程度上是由于无法直接操纵遗传和细胞功能 31。利用定量性状位点 (qtl) 研究确定了调节复杂性状的候选基因, 但这些候选基因的验证一直很困难, 有 32,33, 34。最近, 利用吗啡进行瞬态击倒, 使用 CRISPR 和 T吃饭系统进行基因编辑, 以及使用Tol2介导的转基因技术, 研究了一些性状35、36、37的遗传基础.,38。这些技术的实施和标准化将允许对生物特性的分子和神经基础进行操作, 包括基因功能的操纵、已定义细胞群的标记以及功能记者的表达。虽然这些基因工具的成功实施, 以操纵基因或细胞功能已在紧急模型系统中证明, 详细的协议仍然缺乏在a. mexicanus。
Mexicanus提供了对进化机制的关键洞察, 以应对不断变化的环境, 并提供了识别调节不同性状的新基因的机会。一些因素表明, mexicanus是一个极其可操作的模型, 用于应用现有遗传模型中现有的既定基因组工具, 包括在实验室中轻松维持鱼类的能力、大的育苗大小、透明度, 一个测序的基因组, 并定义行为检测39。在这里, 我们描述了一种方法, 用于使用吗啡, 转基因, 和基因编辑的表面和洞穴种群的. 这些工具在马西卡努斯的更广泛应用将允许对海鱼和河豚发育、生理和行为差异演变背后的分子过程进行机械研究。

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manipulation of gene function in mexican cavefish

洞穴动物提供了一个引人注目的系统, 用于调查许多复杂特征的进化机制和遗传基础, 包括眼睛退化、白化病、睡眠不足、高血糖和感官处理。由于不同洞穴系统之间的环境压力, 来自世界各地的紫鱼物种表现出形态和行为特征的收敛进化。在实验室环境中研究了不同的洞穴物种。墨西哥的泰拉,具有视觉和盲目性的形式, 提供了独特的见解, 生物和分子过程的基础, 进化复杂的性状, 并作为一个新兴的模型系统的良好的准备。虽然在 mexicanus 中已经确定了调节不同生物过程进化的候选基因, 但验证单个基因作用的能力有限。转基因和基因编辑技术的应用有可能克服这一重大障碍, 并研究复杂性状进化的机制。在这里, 我们描述了一种不同的方法来操纵基因表达在 a. mexicanus。方法包括使用吗啡、 Tol2转基因和基因编辑系统来操纵马塞卡努斯的基因功能, 这些系统通常用于斑马鱼和其他鱼类模型. 这些协议包括详细描述定时繁殖程序、受精卵的收集、注射和转基因动物的选择。这些方法将允许研究遗传和神经机制的演变的不同性状的影响。

多种群的血管居住 a . mexicanus显示减少睡眠和增加清醒活性相对于他们的表面居住的康特14。低聚体/奥氏症 (hcrt) 是一种高度保守的神经肽, 其作用是增加清醒, hcrt 途径中的畸变在人类和其他哺乳动物中引起嗜睡症 47,48。我们之前已经证明, 洞穴a. mexicanus增加了 hcrt 肽的表达, 这表明这种肽的表达增加可能是导致睡眠丧失的...

Watch this Scientific Journal Video about Manipulation of Gene Function in Mexican Cavefish at JoVE.com

Manipulation of Gene Function in Mexican Cavefish

我们描述了在进化模型系统中操纵基因的方法. 描述了三种不同的技术: Tol2 介导的转基因、使用 CRISPR/Cas9 对基因组进行有针对性的操纵以及使用吗啡酮敲除表达。这些工具应有助于直接研究表面和洞穴居住形式之间变异背后的基因。

墨西哥鱼基因功能的操纵

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

洞穴动物为研究各种形态、发育和行为特征提供了一个令人信服的系统。但是，建立这个模型系统的限制因素之一是缺乏基因工具，使我们能够操纵基因功能。在这里，我们演示了三种不同的方法来操纵墨西哥洞穴鱼，Astyanax墨西哥人的基因功能。这些工具将有助于研究地表鱼和洞穴鱼之间自然变异的基因，以及它们与表型的关系。贝萨尼·斯塔尔，一个在我的实验室里才华横溢的博士后，将证明这个程序。在开始手术之前，将100毫升的培养皿中溶解在鱼系统水中的温3%agarose，并小心地将蛋注模具以45度角放入加糖中，然后慢慢将霉菌放入加糖中，以避免将空气困在模具下。当加糖凝固后，小心地去除模具，然后在四摄氏度下存放板材长达一周。然后根据制造商的指南，从硅酸盐玻璃毛细管和电极针拉拔器中拔出针头。注射当天，使用玻璃移液器将单细胞卵子转移到注射板中，每排填充多达五行注射板的鸡蛋，每排30至40个鸡蛋。用少量的鱼系统水保持鸡蛋水合。在冰上解冻Morpholino，并准备注射溶液，以便使用长微加载器移液器尖端或向末端添加2至4微升玻利瓦尔，每个鸡蛋注射400个菲莫利诺的象形图。当所有针头都已加载后，使用钳子修剪注射针尖的多余的长度，然后将第一根针头安装到连接到皮莫利特微注射器的微操纵器中。然后，将注射盘放在解剖显微镜下，使用微操纵器用针头穿透每个鸡蛋，然后将一纳米升的Morpholino注射溶液直接注射到蛋黄中。对于CRISPR注射，将引导RNA的25个象形图与300个CRISPR相关蛋白9信使RNA的象形图混合，并如所证明，将产生的RNA溶液的两纳米光注入每个胚胎中。对于 Tol2 转置酶和 Tol2 侧翼质粒注射，将 Tol2 信使RNA每微升 25 纳米图与 Tol2 质粒构造的微升 25 纳米图和无 RNase 水中的酚红色相结合。然后像刚刚证明的，将一纳米升溶液注入每个胚胎。在注射后四天内对莫菲诺注射的动物进行筛查，根据感兴趣的基因，在立体显微镜下对鱼幼虫进行可视化，以观察注射动物的表型或视频记录，以测量行为。要筛选CRISPR内德尔，将CRISPR注射的胚胎移植到PCR管中，然后根据标准DNA提取方案提取DNA，用于聚合酶链反应或PCR分析。要评估诱变，在 70 伏特的 3%agarose 凝胶上运行 PCR 产品的 5 微升，运行 3 小时。野生型、非突变性DNA将产生一个不同的波段，而突变DNA则会导致污迹带。与注入炒莫罗诺的水面鱼相比，注射了对照莫菲诺的墨西哥鱼在24小时内表现出更多的运动活动，睡眠减少。与控制注射的鱼相比，低胰蛋白奥里诺的注射对表面鱼的睡眠影响很小。相比之下，通过Morpholino注射的低克雷素或毒素的敲击对洞穴栖息鱼类的睡眠有显著影响，在低克雷素奥林表达和睡眠损失之间提供了直接的联系。表面鱼类中白化病基因OCA2的CRPSR中化突变导致野生型OCA2阳性等位基因的一些个体同源，并且与携带色素沉着有关，而拥有两个CRISPR突变OCA2阴性等位基因的个体是白化病，在洞穴鱼中突变的OCA2基因和白化病之间提供了直接的联系。选择 F0 的正向，用于 Tol2 传输的转基因回交叉到野生类型，以生成稳定的 F1 线。这支持活钙成像，以揭示神经元活动的差异，调解洞穴环境中的行为变化，为许多其他转基因的表达奠定基础，以描述和操纵墨西哥的基因功能。要记住的最重要的事情是将鸡蛋紧紧地装在盘子上，正确修剪针头，并优化微注射。在成功的基因敲打、转基因整合或CRISPR中继的基因编辑后，这些鱼可用于发育、行为和大脑活动分析。

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Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

科研

遗传学

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

无基因克隆的基因破坏的变形链球菌菌株的生成

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

在秀丽线虫中对倍体的操控

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Culturing and Manipulation of O9-1 Neural Crest Cells

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.

O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing.

O9-1 神经嵴细胞的培养与调控

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and ...

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

提高墨西哥龙舌兰对幼虫后表型和全体免疫组织化学的分析

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...