Os autores gostariam de agradecer ao Dr. M. Cliona McHale pela leitura crítica e edição do manuscrito. Este trabalho foi financiado pelo Instituto Nacional de Ciências de Saúde Ambiental, Instituto Nacional de Saúde para concessão R01ES017452 L Zhang.

1. Preparação da lâmina de amostra <ul><li> FISH OctoChrome é projetado para examinar as alterações cromossômicas em spreads metafásicos de células humanas, incluindo, mas não se limitando a culturas de células de sangue periférico, estaminais / progenitoras células, e linhas celulares. As amostras devem ser preparado seguindo o procedimento padrão para a colheita metáfases 3,11-13: através do emprego de tratamento Colcemid para prender células em metáfase, o tratamento hipotónico a inchar células e, em fixador de Carnoy para consertar células em suspensão em aproximadamente 0,5-1 x 10 6 células por ml. </li><li> Limpe o slide 8-quadrado (fornecido no Kit OctoChrome, Catálogo #: PMP 803, Cytocell Ltd, Cambridge, Reino Unido, a Figura 2) por meio de imersão em etanol puro para 2 min. </li><li> Antes de fazer o slide de amostra, deixe cair uma pequena quantidade de células em uma corrediça de para verificar a densidade de célula. Se a densidade de célula é demasiado elevado (demasiado muitas células sobrepostas), diluir a suspensão com fixador fresco. Se a densidade de célula é demasiado baixo (célula demasiado poucoss em slide), girar as células e re-suspensão em um volume menor de fixador fresco. Pipetar uL 5-10 de suspensão de células para cada uma das 8 áreas de o slide modelo em uma seqüência de alternando quadrados 1/7 - 2/8 - 3/5 - 4/6. Uma vez que os primeiros dois quadrados tem ar-dried, manchar os quadrados restantes na mesma maneira. Isto irá prevenir os spreads celulares a partir de interferindo uns com os outros. </li><li> O slide desidratada pode ser armazenada a -20 ° C em atmosfera de azoto durante maior do que 5 anos antes de hibridação. </li></ul> 2. Localisation de metafases usando um sistema automatizado <ul><li> Aplicar 20 uL de 4 &#39;,6-diamidino-2-fenilindol (0,2 ug / ml) para o centro de cada meia do slide e, em seguida, cobrir com uma lamela. </li><li> Executar uma varredura automática do slide de exemplo para localizar as células em metafase, usando Metafer software (MetaSystems, Altlussheim, Alemanha). Isso facilita a localização de todas as metáfases e futuras re-avaliação de todas as imagens das células. </li><li> Ver os resultados da verificação manualmente e excluir não-metáfase artefatos. </li></ul> 3. Hibridização <ol><li> Preparação da lâmina de amostra e dispositivo de OctoChrome </li><ul><li> Mergulhe o slide amostra em 2x buffer de SSC à temperatura ambiente durante 30 min para lavar afastado DAPI. </li><li> Desidratar o slide amostra através de uma série de lavagens etanol (2 minutos cada, em 70%, 85% e de etanol 100%), permitir que para secar e coloque sobre um 37 ° placa de aquecimento C durante 5 minutos. </li><li> Coloque o Chromoprobe Secção de Hibridização Multiprobe (fornecido no Kit de OctoChrome) em um 37 banho de água ° C e deixar equilibrar à 37 ° C (+ / - 1 ° C). </li><li> Misture a solução de hibridação (fornecido no Kit de OctoChrome) por pipetagem repetida e pré-morna alíquota um 25 uL por dispositivo OctoChrome para 37 ° C. </li><li> Pré-morna dispositivo de cada OctoChrome (fornecido no Kit de OctoChrome, veja a Figura 2) para 37 ° C, colocando o lado da etiqueta dispositivo para baixo sobre uma placa de aquecimento. NÃO toque tele superfícies em alto relevo do dispositivo de OctoChrome como eles contêm as sondas. </li></ul><li> Posicionamento do slide amostra sobre o dispositivo OctoChrome (Figura 2) </li><ul><li> Adicionar 2 uL de pré-aquecida solução de hibridação a cada uma das oito áreas sobre o dispositivo de pré-aquecida OctoChrome enquanto ele permanece a 37 ° C. </li><li> Cuidadosamente, inverta o slide modelo em cima de o dispositivo de OctoChrome tal que o número 1, que é agora de cabeça para baixo, está localizado sobre a área de mão topo direito do dispositivo OctoChrome. Para ajudar a localizar quadrado 1, a sua posição sobre o dispositivo tem sido marcado em Orange. </li><li> Certifique-se de que o slide de modelo é cuidadosamente alinhadas com as áreas correspondentes no dispositivo de OctoChrome. Cuidadosamente abaixar o slide sobre o dispositivo OctoChrome de modo a que as gotas de solução de hibridação fazer contato com o slide. Aplicar gentil, até mesmo de pressão para garantir que a solução de hibridação é se espalhar para as bordas de cada uma das áreas levantadas sobre o dispositivo OctoChrome. </li><li> Levantaro slide / OctoChrome, cuidadosamente segurando o fim geado da lâmina de vidro, e inverter de modo que o slide é por baixo do aparelho OctoChrome. Verifique se o dispositivo não mancha em todo o slide modelo, pois isso pode causar a contaminação cruzada das sondas. </li><li> Colocá-lo sobre um 37 ° C (+ / - 1 ° C) placa de aquecimento durante 10 minutos. </li></ul><li> Desnaturação </li><ul><li> Transferir a amostra dispositivo slide / OctoChrome para a placa de aquecimento tendo o cuidado especial para mantê-lo nivelado. Verifique se o slide amostra uniformemente contatos da placa. </li><li> Desnaturar sobre a placa de aquecimento, a 75 ° C (+ / - 1 ° C) por 5 minutos. </li></ul><li> Hibridização </li><ul><li> Local de slides amostra / dispositivo OctoChrome na Câmara de Hibridização Chromoprobe Multiprobe. </li><li> Substitua a tampa e flutuar da câmara em o 37 ° C (+ / - 1 ° C) banho de água (não - de agitação) durante a noite. Atenção: não fechar a tampa da câmara de hibridação; não fechar a tampa do banho de água; NÃO hibridizar em umincubadora. Essas etapas são críticas para controlando umidade. </li></ul><li> Pós-hibridação lavagens </li><ul><li> Preparação de soluções de lavagem Solução 1: Prepare uma jarra de Coplin / Hellendahl contendo 0.4x SSC. Equilibre com a 72 ° C (+ / - 1 ° C) em um banho de água e ajustar o pH a 7,0. Solução 2: Prepare a uma jarra de Coplin / Hellendahl contendo 2x SSC e 0,05% de Tween 20. Permitir que a repousar à temperatura de quarto. </li><li> Remova o dispositivo OctoChrome cuidadosamente a partir do slide e coloque o slide no Solution 1 por 2 minutos. </li><li> Coloque o slide em Solução 2 por 30 segundos. </li></ul></ol> 4. Montagem e visualização de resultados <ul><li> Aplicar 20 uL de DAPI para o centro de cada meia do slide e aplicar uma lamela. </li><li> Incubar no escuro, durante 10 minutos à temperatura ambiente antes de visualizar por microscopia de fluorescência. </li></ul> 5. Os resultados representativos FIGUre 3A mostra uma célula de metáfase normal de na Praça de 2. (1) - (3): Os cromossomos 8, 21 e 12 foram pintadas de vermelho, verde e azul, e são visualizados através de um Red Texas, FITC, e filtro DEAC, respectivamente; (4): A visualização através de um DAPI / FITC / Texas Red triplo filtro. Geralmente, os cromossomos pintadas com azul não são claras através do filtro triplo e precisam de ser encarados sob o filtro DEAC específico. Figura 3B mostra representativas células anormais com leucemia-específico translocação cromossômica e aneuploidias. (1): t (8; 21), uma translocação cromossômica comum na leucemia mielóide aguda, (2): Trissomia 21, três cópias do cromossomo 21, a aneuploidia comum na leucemia. <img alt="Figura 1" src="/files/ftp_upload/3619/3619fig1.jpg" /> Figura 1. Arranjo de combinações de cromossomos no dispositivo OctoChrome e resultados esperados pintura cromossômicas. <img alt="Figura 2" src= &quot;/ Files/ftp_upload/3619/3619fig2.jpg&quot; /&gt; Figura 2. De Posicionamento da lâmina de poços amostra sobre o dispositivo OctoChrome. Figura 3. Os resultados representativosobtido a partir de FISH OctoChrome (Praça 2). <img alt="Figura 3A" src="/files/ftp_upload/3619/3619fig3A.jpg" /> Figura 3A. Uma célula normal com cromossomos 8, 12, 21 pintados na Praça 2. <img alt="Figura 3B" src="/files/ftp_upload/3619/3619fig3B.jpg" /> Figura 3B. Representante células anormais com leucemia-específico translocação cromossômica e aneuploidias na Praça 2.

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Este teste FISH-OctoChrome romance permite, simultaneamente, examinar todos os 24 cromossomos humanos em uma hibridação única em um slide. O arranjo de combinações cromossómicas sobre os 8 quadrados foi projetado para facilitar a identificação de os rearranjos não-aleatórios cromossómicas estruturais nas leucemias mais comuns e linfomas. Assim, o ensaio pode detectar numérica (aneuploidia), bem como estruturais alterações (translocações) cromossómicas concorrentemente. O pas...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td><tr><tr><td> OctoChrome Kit </td><td> Cytocell Ltd, Cambridge, Reino Unido </td><td> PMP 803 </td><td></td></tr><tr><td> fitohemaglutinina (PHA) </td><td> Invitrogen Corporation </td><td> 10576-015 </td><td></td></tr><tr><td> Colcemid </td><td> Invitrogen Corporation </td><td> 15212-012 </td><td></td></tr><tr><td> Fixador de Carnoy </td><td></td><td></td><td> Metanol: ácido acético glacial = 3: 1 </td></tr><tr><td> Fluorescência microscope </td><td></td><td></td><td> Equipado com filtros para visualizar DAPI, Texas Red, FITC, eo espectro de cumarina individualmente e DAPI / FITC / Texas Red triplo filtro para visualizar as cores diferentes simultaneamente </td></tr><tr><td> Metafer software </td><td> MetaSystems, Altlussheim, Alemanha </td><td></td><td> Facilite para localizar todas as metáfases e re-avaliar as anormalidades </td></tr></tbody></table>

chromosomics: detection of numerical and structural alterations in all 24 human chromosomes simultaneously using a novel octochrome fish assay

De fluorescência de hibridização in situ (FISH) é uma técnica que permite que seqüências específicas de DNA a ser detectado em cromossomos metafásicos ou de interfase em núcleos de células 1. A técnica utiliza sondas de DNA com sequências únicas que hibridizam a cromossomos inteiros ou específicas regiões cromossômicas, e serve como um adjunct poderosa para citogenética clássicos. Por exemplo, muitos estudos anteriores relataram a ocorrência freqüente de aberrações cromossômicas maiores em pacientes com leucemia relacionados com exposição ao benzeno, benzeno envenenamento de pacientes e trabalhadores saudáveis ​​expostos ao benzeno, utilizando análise citogenética clássica 2. Usando FISH, leucemia específicos alterações cromossômicas foram observadas a ser elevados em trabalhadores aparentemente saudáveis ​​expostos ao benzeno 3-6, indicando os papéis críticos de mudanças citogênicas em benzeno induzida leucemogênese. Geralmente, um teste FISH único examina apenas um ou poucos cromossomos inteirosou loci específicos por slide, etc hibridizações múltiplas precisam ser realizados em vários slides para cobrir todos os cromossomos humanos. Cariotipagem espectral (SKY) permite a visualização de todo o genoma simultaneamente, mas a necessidade de software especial e limitações do equipamento a sua aplicação 7. Aqui, descrevemos um teste FISH romance, OctoChrome FISH, que pode ser aplicado para Chromosomics, que definimos aqui como a análise simultânea de todos os 24 cromossomos humanos em um slide em estudos humanos, tais como estudo de todo o cromossomo aneuploidia (CWA) 8. A base do método, comercializado pela Cytocell como o Sistema de Multiprobe Chromoprobe, é um dispositivo de OctoChrome que está dividido em 8 quadrados, contendo cada um dos quais transporta três diferentes sondas cromossômicas inteiras de pintura (Figura 1). Cada uma das três sondas está diretamente rotulado com um fluoróforo colorido diferente, verde (FITC), vermelho (Texas Red), e azul (A cumarina). O arranjo de combinações cromossómicas sobre a OctoChrome dispositivo foi projetado para facilitar a identificação das não-aleatórias alterações cromossômicas estruturais (translocações) encontrados nas leucemias e linfomas mais comuns, por exemplo t (9; 22), t (15; 17), t (8; 21 ), t (14; 18) 9. Além disso, mudanças numéricas (aneuploidia) em cromossomos pode ser detectada concorrentemente. O slide de modelo correspondente também é dividido em 8 quadrados sobre a qual spreads em metafase são vinculados (Figura 2), e é posicionado sobre o dispositivo de OctoChrome. As sondas e DNA-alvo são desnaturado a alta-temperatura e hibridizadas em uma câmara úmida, e, em seguida, todos os 24 cromossomos humanos podem ser visualizados simultaneamente. FISH OctoChrome é uma técnica promissora para o diagnóstico clínico de leucemia e linfoma e para a detecção de aneuploidias em todos os cromossomos. Nós aplicamos esta nova abordagem cromossômica em um estudo CWA de benzeno expostos os trabalhadores chineses 8,10.

Watch this Scientific Journal Video about Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay at JoVE.com

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Um de fluorescência romance<em&gt; In situ</em&gt; Hibridização (FISH) método que, simultaneamente, examina alterações cromossômicas, tanto numéricas e estruturais, particularmente as translocações específicas cromossômicas associados com leucemia e linfoma, de todos os 24 cromossomos humanos em um único dispositivo em uma hibridação, é descrito.

Chromosomics: Detecção de alterações numéricas e estruturais em todos os 24 cromossomos humanos ao mesmo tempo usando um ensaio de FISH Novel OctoChrome

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

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JoVE Journal

Biology

18.7K visualizações.  University of California, Berkeley. Um de fluorescência romance In situ Hibridização (FISH) método que, simultaneamente, examina alterações cromossômicas, tanto numéricas e estruturais, particularmente as translocações específicas cromossômicas associados com leucemia e linfoma, de todos os 24 cromossomos humanos em um único dispositivo em uma hibridação, é descrito.

Vídeo: Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Fluorescência In situ Hybridization (FISH) Protocolo no esperma humano

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Biologia

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

Detecção de Sinalização de Efeitos Complexos Downstream de BMP4 Usando In situ PLA, um Ensaio Ligadura Proximidade

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Alto rendimento Assay Sacarificação para materiais lignocelulósicos

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Detecção de RNA viral por fluorescência<em&gt; In situ</em&gt; Hybridization (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Alta capacidade mapeamento físico de cromossomos usando Automated<em&gt; In situ</em&gt; Hibridação

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Detectando somáticas Genéticos Alterações em amostras de tumor por Exon Captura e Seqüenciamento Massively Parallel

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Produção eficiente e Purificação de recombinante murino Kindlin-3 a partir de células de insetos de Estudos biofísicos

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, 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.

Avaliando Contribuições Espécies específicas Para o Desenvolvimento Craniofacial Utilizando Quimeras Codorniz-pato

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

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

Expressão de proteínas recombinantes de Biologia Estrutural em HEK 293F Suspensão Cells: Uma abordagem inovadora e acessível

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

Detecção de intracelular Expressão Gênica em células vivas de murino, humano e porcino Origem Usando nanopartículas marcados com fluorescência

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Methods Article

Chromosomics: Detecção de alterações numéricas e estruturais em todos os 24 cromossomos humanos ao mesmo tempo usando um ensaio de FISH Novel OctoChrome