这项工作得到了在儿童探知研究所授予的MC-二-2006年-1（RDM的和泰德），在NIH的表观遗传学蓝图补助金[1R01DA025744-01和3R01DA025744-02S1]（RDM并且FLMV），U01AG023746（SC）的，在Saigh基金会（FLMV和TED），1K08CA140720-01A1和亚历克斯的柠檬水摊位"一奖"支持（TED）。我们感谢与基因组分析的帮助下，在华盛顿大学医学院遗传学系的基因组技术访问中心。该中心的部分支持NCI癌症中心支援津贴＃P30的CA91842 Siteman癌症中心和ICTS /铁协批准UL1RR024992从NationalCenter研究资源（NCRR），美国国立卫生研究院（NIH）的一个组成部分，并国立卫生研究院医学研究路线图。本刊物完全是作者的责任，并不代表NCRR或NIH的官方意见。

<ol>
	<li>Vallania, F. L. M., Druley, T. E., Ramos, E., Wang, J., Borecki, I., Province, M., Mitra, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+discovery+of+rare+insertions+and+deletions+in+large+cohorts.">High-throughput discovery of rare insertions and deletions in large cohorts.</a> Genome Research. 20, 1391-1397 (2010).</li><li>Shendure, J., Mitra, R., Varma, C., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+Sequencing+Technologies:+Methods+and+Goals.">Advanced Sequencing Technologies: Methods and Goals.</a> Nature Reviews of Genetics. 5, 335-344 (2004).</li><li>The 1000 Genomes Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+map+of+human+genome+variation+from+population-scale+sequencing.">A map of human genome variation from population-scale sequencing.</a> Nature. 467, 1061-1073 (2010).</li><li>Manolio, T. A., Collins, F. S., Cox, N. J., Goldstein, D. B., Hindorff, L. A., Hunter, D. J., McCarthy, M. I., Ramos, E. M., Cardon, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+the+missing+heritability+of+complex+diseases.">Finding the missing heritability of complex diseases.</a> Nature. 461, 747-753 (2009).</li><li>Reich, D. E., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+allelic+spectrum+of+human+disease.">On the allelic spectrum of human disease.</a> Trends Genet. 17, 502-510 (2001).</li><li>Cohen, J. C., Kiss, R. S., Pertsemlidis, A., Marcel, Y. L., McPherson, R., Hobbs, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+rare+alleles+contribute+to+low+plasma+levels+of+HDL+cholesterol.">Multiple rare alleles contribute to low plasma levels of HDL cholesterol.</a> Science. 305, 869-872 (2004).</li><li>Ji, W., Foo, J. N., O'Roak, B. J., Zhao, H., Larson, M. G., Simon, D. B., Newton-Cheh, C., State, M. W., Levy, D., Lifton, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rare+independent+mutations+in+renal+salt+handling+genes+contribute+to+blood+pressure+variation.">Rare independent mutations in renal salt handling genes contribute to blood pressure variation.</a> Nat. Genet. 40, 592-599 (2008).</li><li>Ahituv, N., Kavaslar, N., Schackwitz, W., Ustaszewska, A., Martin, J., Hebert, S., Doelle, H., Ersoy, B., Kryukov, G., Schmidt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+sequencing+at+the+extremes+of+human+body+mass.">Medical sequencing at the extremes of human body mass.</a> Am. J. Hum. Genet. 80, 779-791 (2007).</li><li>Druley, T. E., Vallania, F. L., Wegner, D. J., Varley, K. E., Knowles, O. L., Bonds, J. A., Robison, S. W., Doniger, S. W., Hamvas, A., Cole, F. S., Fay, J. C., Mitra, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+rare+allelic+variants+from+pooled+genomic+DNA.">Quantification of rare allelic variants from pooled genomic DNA.</a> Nat. Methods. 6, 263-265 (2009).</li><li>Mitra, R. D., Butty, V., Shendure, J., Housman, D., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+Genotyping+and+Haplotyping+with+Polymerase+Colonies.">Digital Genotyping and Haplotyping with Polymerase Colonies.</a> Proc. Natl. Acad. Sci. 100, 5926-5931 (2003).</li></ol>

没有利益冲突的声明。

有越来越多的证据表明，发病率和治疗反应常见的，复杂的表型和8肥胖，高胆固醇血症，高血压7和其他疾病，如可由个人档案罕见的变异主持。确定基因和途径，这些变种在受影响的人口聚集的地方将产生深远的诊断和治疗的影响，但单独分析受影响的个人，可以是时间和成本过高。人口为基础的分析提供了一个更有效的方法测量在多个位点的遗传变异。 我们提出一个新的池DNA测序协议旨在确定这个种群之间的遗传变异类型的​​与斯普林特软件的包配对。我们证明这种方法的准确性，在确定和量化次要等位基因在947个人的人口大汇集，包括罕见的变异从头从汇集测序和个人的焦磷酸测序验证。我们的策略主要是从其他协议不同，由一个积极的成立和在每一个实验的阴性对照。这允许分裂，以达到更高的精度和电源相比，其他方法1。每个等位基因的25倍，最佳的覆盖范围是固定的，独立池的大小，大池分析是可行的，这一要求与池大小的唯一尺度线性。我们的做法是非常灵活，可以应用于任何利益的表型，但也自然异构的样品，如混合细胞群和肿瘤活检。由于汇集从大的目标区域，如外显子组或基因组的测序日益增长的兴趣，我们的图书馆准备和碎片分析与自定义捕捉和全外显子组测序兼容，但没有被设计为在分裂包的对齐工具大引用序列。因此，我们已经成功地利用动态规划对准，Novoalign，从汇集的样本（拉莫斯等。提交）调用的变种的全基因组的路线。因此，我们汇集的测序战略可以成功扩展到较大的池，越来越多的靶序列。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 部分 </td></tr><tr><td> PfuUltra高，富达</td><td>安捷伦</td><td> 600384 </td><td> 1.4 </td></tr><tr><td>甜菜碱</td><td>西格玛</td><td> B2629 </td><td> 1.4 </td></tr><tr><td> m13mp18单链DNA载体</td><td> NEB </td><td> N4040S </td><td> 1.5 </td></tr><tr><td>至pGEM-T容易</td><td> Promega公司</td><td> A1360 </td><td> 1.5 </td></tr><tr><td> T4聚核苷酸激酶</td><td> NEB </td><td> M0201S </td><td> 2.2 </td></tr><tr><td> T4连接酶</td><td> NEB </td><td> M0202S </td><td> 2.2 </td></tr><tr><td>聚乙二醇8000兆瓦</td><td>西格玛</td><td> P5413 </td><td> 2.2 </td></tr><tr><td> bioruptor sonicator </td><td> diagenode </td><td> UCD-200-TS</td><td> 2.3 </td></tr></tbody></table>

detection of rare genomic variants from pooled sequencing using splinter

随着DNA测序技术已经明显提前2近年来，它已成为越来越明显，任何两个人之间的遗传变异量是大于以前认为的3。相比之下，基于阵列的基因分型并没有确定一个共同的序列变异4,5的常见疾病的表型变异的重大贡献。两者合计，这些意见已导致常见疾病的演化/罕见的变异假说，表明多数是"缺失的遗传"中常见的和复杂的表型，而不是由于个人的个人资料罕见的或私人的DNA变异6-8 。然而，表征罕见的变异如何影响复杂表型需要许多受影响的个人，在许多基因位点的分析，是理想到不受影响的队列中的类似调查相比。尽管今天的平台，所提供的电源排序许多基因位点和随后的计算分析所需的人口为基础的调查仍让许多研究者望而却步。 为了满足这一需要，我们开发了汇集的测序方法1,9和一个新的软件包，从得到的数据高度准确的稀有变异检测1。从受影响的个人和整个人口调查的遗传变异程度在多个有针对性的地区，在一个单一的测序库池基因组的能力提供了极好的节约成本和时间，传统的单一样本的测序方法。平均每25倍的基因测序覆盖，我们的自定义算法，分裂，使用内部变量，调用控制策略调用4个碱基对的插入，删除和替换从高灵敏度和特异性的长度可达池1在500个人的突变等位基因。这里，我们描述为准备汇集小号的方法，其次equencing库如何使用汇集测序分析（碎片包一步一步的指示<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a> ）。我们展示了汇集了947个人的测序之间的比较，所有的人也经历了全基因组阵列，超过20KB每人测序。标签的基因分型，并呼吁汇集样品中的新变种之间的一致性都非常优秀。这种方法可以很容易地扩展到任何数量的基因位点和任何个人。通过整合内部的正面和负面的扩增控制模拟研究人口的比率，该算法可以进行校准，以获得最佳性能。这一战略也被修改为与杂交捕获或个别特定条形码的使用，可应用于自然异质性样本，如肿瘤DNA测序。

Watch this Scientific Journal Video about Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER at JoVE.com

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

汇集DNA测序检测与大同伙的复杂表型相关的罕见的变异是一个快速和具有成本效益的战略。在这里，我们描述的汇集，32个癌症相关的基因，利用分裂软件包下一代测序的计算分析。这种方法可扩展性，适用于任何利益型。

罕见的基因变异检测从集资使用斯普林特测序

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

detection-rare-genomic-variants-from-pooled-sequencing-using

Nanyang Technological University

Research

JoVE Journal

Biology

15.1K 次观看.  Washington University School of Medicine. 汇集DNA测序检测与大同伙的复杂表型相关的罕见的变异是一个快速和具有成本效益的战略。在这里，我们描述的汇集，32个癌症相关的基因，利用分裂软件包下一代测序的计算分析。这种方法可扩展性，适用于任何利益型。

视频: Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

科研

生物学

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

超高分子量基因组DNA的提取土壤和沉积物

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

磁共振成像基因 - 公共资源为研究基因组DNA序列内模式

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

研究年龄相关的基因组不稳定性使用 S。酵母年代的寿命模型

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

阵列比较基因组杂交（CGH阵列），用于检测基因拷贝数变异的

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

隔离增效变种的Hsp104利用酵母Proteinopathy模型

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

斑马鱼肾功能评价采用荧光测定间隙

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.

TRAP-RC，翻译核糖体亲和纯化的稀有细胞群<em&gt;果蝇</em&gt;胚胎

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given ...