Esta pesquisa foi suportada por subvenção R01NS062842 do Instituto Nacional de Distúrbios Neurológicos e Derrame, National Institutes of Health (VVD) e por doações R21 NS064403 do Instituto Nacional de Distúrbios Neurológicos e Derrame, National Institutes of Health através ARRA (VVD) e R21 EB006301 National Institute of Biomedical Imaging e Bioengenharia, National Institutes of Health (VVD).

As seções para a detecção baseada em máquina molecular deve ser preparado em primeiro lugar porque a sua preparação leva mais tempo do que a montagem do dispositivo molecular. A construção funciona bem com 5-6μm de espessura, corte de paraformaldeído fixo, blocos de parafina do tecido. Use marcas de slides que retêm seções bem, como ProbeOn Além disso slides carregada e precleaned (Fisher Scientific) ou similar. Recomendamos em primeiro lugar usando um tecido com um padrão conhecido de dano ao DNA, que contém tanto DNase I e tipo II DNase-breaks, como a dexametasona tratadas com ratos apoptóticas timo 13,14. 1. Preparação de seções <ol><li> Coloque as seções em um rack de slides e dewax em xilol por 15 min, transferir para um banho de xileno fresco por mais 5 minutos. </li><li> Reidratar passando por concentrações de etanol classificados: 96% de etanol 2x5min; Etanol 80% - 5min; água - 2x5 min. </li><li> Digest seção com Proteínase K. Use 100μL de uma solução de 50μg/mL por seção. Incubar 15 &#39;à temperatura ambiente (23 ° C) em câmara úmida. O tempo pode precisar de ajustes, dependendo do tipo de tecido. Tecidos duros pode exigir mais tempo de digestão. Vezes de 15-25 min são normalmente utilizados. Digestão insuficiente pode resultar no sinal mais fraco. Overdigestion na mão resulta no desaparecimento de sinais e interrupção seção. </li><li> Enxágüe em água destilada por min 2x10. </li><li> Adicionar 100 mL por seção da BSA 2% para preblocking. Incubar por 15 min à temperatura ambiente (23 ° C). Durante esse tempo, montar a máquina molecular. </li></ol> 2. Montagem máquinas moleculares Todos os reagentes são escalados para 25 mL de volume total, que é suficiente para uma detecção única em uma seção média dimensão do tecido (10x10mm). O volume pode ser ampliado conforme a necessidade. <ol><li> Combine em um pequeno tubo de plástico na seguinte ordem: </li></ol><table border="1"><tbody> &lt;tr&gt; <td><ol><li> Água bidestilada; </li><li> Glicol-8000 de polietileno de 15%; </li><li> Solução de 66 mM Tris-HCl, pH 7,5, 5 mM MgCl 2, 0,1 mM Ditioeritritol, 1 mM ATP, (ie tampão ligase regulares T4 DNA); </li><li> 70 pmoles de Oligonucleotídeos 1, </li><li> 215 pmoles (1,76-7,1 mg) VACC TOPO </li><li> 10 unidades de T4 DNA ligase (500 U / mL) </li></ol></td></tr></tbody></table><ol start="2"><li> Misture suavemente por pipetagem. A construção molecular quase instantaneamente auto-monta nesta solução e pode ser usado imediatamente em temperatura ambiente (23 ° C). Aumentando a temperatura para 37 ° C bloqueia a T4 DNA ligase componente baseado em matéria de rotulagem. </li></ol> 3. Utilização de máquinas moleculares em seções de tecido para 5&#39;OH etiqueta dupla e 5&#39;PO4 breaks DNA <ol><li> Aspirar a solução preblocking e aplicar 25μL da mistura de reacção completa contendo 70 pmoles de Oligonucleotídeos 1, 53-215 pmoles (1,76-7,1 mg) VACCTOPO e 10 unidades de T4 DNA ligase (500 U / mL) em solução de 66 mM Tris-HCl, pH 7,5, MgCl 5mM 2, 0,1 mM Ditioeritritol, 1 mM ATP, e polietileno glicol 15%-8000. O oligonucleotídeo mesma sequência carregando um único rótulo FITC, Oligonucleotide 2, pode ser usado para a detecção em vez de um único tipo de quebras de DNA. Em tal caso, omitir ligase a partir da reação rotulagem. Também nesta situação uma solução de 50 mM Tris-HCL, pH 7,4, 15% PEG-8000 pode ser usado em vez de tampão T4 DNA ligase. </li><li> Incubar por 1 hora em temperatura ambiente (23 ° C) em câmara úmida com uma lamela de plástico. Proteger da luz. Diminuição da temperatura para 16 ° C reduz o sinal ligase baseado, o aumento da temperatura a 37 ° C elimina completamente o sinal ligase-based. A inibição parcial da ligase às vezes ocorre na mistura de reação, possivelmente devido a contaminantes introduzidos com os preparativos topoisomerase. Portanto, mais de incubação (2-4 horas) pode ser necessária, especialmente quando snúmeros ignificant de ligase marcado breaks estão presentes. Embora ambos os ligase e sinais de topo pode ser observado nesta fase, em muitos casos o sinal ligase pode ser reforçada pela re-aplicação da mistura de reação sem VACC TOPO e Oligonucleotide dual-rotulados, mas contendo um oligonucleotídeo forma hairpin (Oligonucleotide 3) (35 mg / mL) e T4 DNA ligase (250 U / mL). Para melhorar sinal vá para a Etapa 3, para ver a reação, sem realce vá para o Passo 5. </li><li> Remove delicadamente lamínulas imergindo as lâminas verticalmente em uma jarra de Coplin contendo água à temperatura ambiente. Aspirar o excesso de água. </li><li> Melhorar sinal ligase através da aplicação de 25 mL de mistura de reação sem VACC TOPO e Oligonucleotide 1, mas contendo Oligonucleotide 3: 66 mM Tris-HCl, pH 7,5, 5 mM MgCl 2, 0,1 mM Ditioeritritol, 1 mM ATP, e polietileno glicol 15% 8000, 5 unidades de T4 DNA ligase, 35 de Oligonucleotídeos mg / mL 3 (blunt hairpin terminou). O volume total da solução de rotulagem can ser ampliados para acomodar as seções maiores. Incubar por 18 horas (overnight) em temperatura ambiente (23 ° C) em câmara úmida com uma lamela de plástico. </li><li> Remove delicadamente lamínulas imergindo as lâminas verticalmente em Coplin jar contendo água à temperatura ambiente. Em seguida, lave min seção 3x10 em água destilada. </li><li> Enxaguar com tampão bicarbonato de sódio. Solução alcalina lavar aumenta fluorescência FITC, que é sensível e pH é significativamente reduzido em pH abaixo de 7. </li><li> Cobrir seção com uma solução antifading (Vectashield com DAPI), lamela e analisar o sinal usando um microscópio de fluorescência. Quebra dupla vertente-DNA com 5&#39;OH ficam verde-fluorescentes, 5&#39;PO quatro breaks - vai fluorescência vermelha (Figura 2). </li></ol> 4. Resultados representante <img alt="Figura 2." src="/files/ftp_upload/3628/3628fig2.jpg" /> Figura 1. Semi-artificial máquina molecular detecta dois tipos deDanos ao DNA in situ. A máquina de auto-fluorescente quando monta VACC TOPO liga-se à dupla hairpin-32-mer. A máquina começa a operação, dividindo-se em duas unidades detector via topoisomerase-made corte na extremidade 3 &#39;da seqüência de reconhecimento. Isso resulta em um processo cíclico onde a unidade de FITC-rotulados de forma contínua separa e volta para o religates rodamina marcado unidade. Este persiste até uma pausa DNA detectável é encontrado. Quando um receptor de tal alternativa (quebra de DNA 5&#39;OH blunt end) está presente na seção de tecido, a parte FITC vai ligadura a ele. A parte restante rodamina serão anexados a 5 &#39;PO 4 DNA quebra extremidade sem corte com a ajuda do DNA ligase T4. Conseqüentemente, ambos os tipos de quebras de DNA são simultaneamente detectado. <img alt="Figura 1." src="/files/ftp_upload/3628/3628fig1.jpg" /> Figura 2. Molecular rótulos máquina dual dois tipos de quebras de DNAem uma seção de tecido de dexametasona tratadas com timo. Blunt quebra-ended DNA de DNase I-II-e DNase tipo são detectados nas áreas cortical do timo em apoptose. Fluorescência citoplasmática verde (5&#39;OH quebra de DNA) marca macrófagos cortical digerir material nuclear de timócitos apoptóticos. Este sinal é produzido por VACC TOPO, e localiza-se fagolisossomos com DNA contendo 5&#39;OH dupla vertente-breaks 11,12. Fluorescência vermelha (5&#39;PO 4 breaks) rótulos de núcleos de timócitos apoptóticos não engolida pelos macrófagos. Um enorme número de timócitos em apoptose simultaneamente acompanhada pela geração de 4 5&#39;PO fita dupla-breaks, visualizado por ligase baseado rotulagem. Estas pausas estão localizadas na periferia nuclear, formando padrões em forma de anel. Todos os núcleos celulares são visualizados através da contracoloração com DAPI (azul fluorescente).

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Neste vídeo, demonstramos como montar e usar um dual-rotulagem sensor de danos ao DNA. O sensor é uma máquina molecular impulsionada por bio-molecular do motor, um vírus proteína codificada TOPO VACC ligados com componentes artificiais. O desenvolvimento apresentado exemplifica uma abordagem bio-enabled que defende a adaptação das estruturas biológicas, arquiteturas e peças e componentes reais de células para o projeto de não-tóxico dispositivos à escala molecular 12,15. Esta abordagem resolve du...

<ol><li> Xileno </li><li> 80 e Etanol a 96%. </li><li> Solução 2% de albumina bovina (BSA) em água destilada. </li><li> Oligonucleotídeo 1: um grampo duplo, dual marcado com fluoresceína e tetrametilrodamina: </li></ol> 5&#39;-GGA AAG CCT GC F GGT GCA CCC ATT ACG CAT R AT GCG TT-3 &#39;; F - FITC-dT; R - tetrametilrodamina-dT. Outros vermelho deslocado fluoróforos, tais como BODIPY TR, rodamina ou TAMRA pode ser usado em vez de R tetrametilrodamina como. Alternativamente, você pode usar 2 Oligonucleotide -. Hairpin uma única dupla marcada com fluoresceína (para a detecção de um único tipo de quebras de DNA (DNase II tipo apenas) A sonda single-fluoróforo de transporte é consideravelmente menos caro e é conveniente sempre que um único tipo de quebra de DNA deve ser rotulado: 5&#39;-GGA AAG CCT GC F GGT GCA CCC ATT ACG CAT ATG CGT T-3 &#39;; F - FITC-dT </p&gt; <ol start="5"><li> Oligonucleotídeo 3. Blunt-ended rodamina marcado hairpin para o realce do sinal de ligadura situ com DNA ligase T4: 5&#39;-GCG CTA GAC C G R GTC TAG CGC-3 &#39;; R - tetrametilrodamina-dT </li><li> Vaccinia DNA topoisomerase l (VACC TOPO) - 3000 U / mL (Vivid Technologies). Nos experimentos iniciais foram utilizados 215 pmoles (7,1 mg) do TOPO VACC por cada mL 25 da mistura de reacção. No entanto, a concentração topoisomerase pode ser significativamente reduzido sem perda de sensibilidade. Mais tarde, usou uma quantidade quatro vezes menor de VACC TOPO por seção (1,76 g em 25 mL da mistura de reação por seção) com resultados semelhantes. Reduzir a quantidade da enzima a 880 ng (em 25 mL da mistura de reação) resultou em um sinal mais fraco e 8,8 ng de VACC TOPO não produziu nenhum sinal detectável. </li><li> DNA ligase T4 5 U / mL (Roche). Esta preparação altamente concentrada ligase dá o melhor sinal em nossas condições experimentais. </li&gt; <li> 10 x tampão de reação para a T4 DNA ligase: 660 mM Tris-HCl, 50 mM MgCl 2, 10 Ditioeritritol mM, 10 mM ATP, pH 7,5 (20 ° C) (Roche). ATP em tampão de reação é facilmente destruído em ciclos repetitivos de descongelamento-congelamento. Alíquota do buffer em pequenas porções 15-20 mL e armazenar a - 20 ° C. Use uma vez. </li><li> 30% (w / v) solução de PEG-8000 (Sigma) em água bidestilada. 15% PEG-8000 na mistura de reacção fortemente estimula a detecção, aumentando as concentrações eficazes dos seus constituintes, por exclusão de volume. </li><li> Proteinase K (Roche) 20 solução estoque mg / mL em água destilada. Armazenar a - 20 ° C. Na solução use reação 50mg/ml em PBS, preparado a partir do estoque. Não reutilize. </li><li> Vectashield com DAPI (Vector Laboratories). </li><li> Tampão de bicarbonato de sódio: 50m M NaHCO 3, 15m M NaCl, pH 8,2. </li><li> Vidro 22x22mm ou 22x40mm ou lamelas de plástico. Lamínulas de plástico são preferíveis durante a reação, já que umre mais fácil de remover da seção. </li></ol>

in vitro assembly of semi-artificial molecular machine and its use for detection of dna damage

Naturalmente bio-molecular máquinas de trabalho em cada célula viva e exibir uma variedade de modelos 06/01. No entanto, o desenvolvimento de centros artificial molecular máquinas em dispositivos capazes de movimento direcional, ou seja, motores moleculares, e sobre a sua escala reduzida partes mecânicas (rodas, eixos, etc pingentes) 7-9. Este imita as máquinas macro-, mesmo que as propriedades físicas essenciais para estes dispositivos, tais como a inércia ea conservação do momento, não são utilizáveis ​​em ambientes nanoworld 10. Projetos alternativos, que não seguem os esquemas mecânicos macromachines e usar mecanismos desenvolvidos na evolução de moléculas biológicas, pode aproveitar as condições específicas do mundo nano. Além disso, adaptando reais de moléculas biológicas para fins de nano-design reduz perigos em potencial dos produtos da nanotecnologia pode representar. Aqui demonstramos a montagem e aplicação de uma construção bio-enabled tais, comoemi-artificial dispositivo molecular que combina uma máquina molecular de ocorrência natural com componentes artificiais. Do ponto de vista enzimologia, a nossa construção é um designer complexo enzima-substrato fluorescente colocados juntos para executar uma função específica útil. Este conjunto é, por definição, uma máquina molecular, uma vez que contém um 12. No entanto, sua integração com a parte de engenharia - hairpin dupla fluorescentes - re-direciona para uma nova tarefa de rotulagem danos no DNA 12. Nossa construção reúne a partir de um DNA 32-mer e uma enzima topoisomerase I vaccinia (VACC TOPO). A máquina então usa seu próprio material para fabricar duas unidades detector fluorescente etiquetado (Figura 1). Uma das unidades (fluorescência verde) carrega VACC TOPO covalentemente ligado a seu 3&#39;end e outra unidade (fluorescência vermelha) é um hairpin livre com uma 3&#39;OH terminal. As unidades são de curta duração e rapidamente montar de volta para a construção original, que posteriormente recleaves.Na ausência de DNA quebra estas duas unidades separadas de forma contínua e religate de uma forma cíclica. Em cortes de tecidos com danos no DNA, a unidade de transporte de detector topoisomerase seletivamente atribui a quebra ponta e DNA com 5&#39;OH (DNase II tipo-breaks) 11,12, fluorescente rotulá-los. O segundo, hairpin enzima livre formado após clivagem oligonucleotídeo, vai a uma ligadura 5&#39;PO 4 quebrar blunt-ended (DNase I tipo-breaks) 11,12, se ligase T4 DNA está presente na solução 13,14. Quando ligase T4 DNA é adicionado a uma secção de tecido ou uma solução contendo DNA com 4 5&#39;PO ponta e breaks, a ligase reage com 5&#39;PO quatro extremidades do DNA, formando semi-estável da enzima DNA-complexos. Os grampos de cabelo sem corte terminou irá interagir com estes complexos liberando hairpins ligase e covalentemente ligados a DNA, assim rotulagem 5&#39;PO quatro breaks ponta e DNA. Este desenvolvimento exemplifica uma nova abordagem prática para podesign e de máquinas moleculares e fornece um sensor de útil para a detecção de apoptose e dano de DNA em células fixas e tecidos.

Watch this Scientific Journal Video about In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage at JoVE.com

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Nós demonstramos a montagem e aplicação de um dispositivo em escala molecular alimentado por uma proteína topoisomerase. A construção é um sensor de bio-molecular que rotula dois principais tipos de quebras de DNA em cortes de tecido, anexando dois fluoróforos diferentes para os seus fins.

Em Assembléia in vitro de Semi-artificial máquina molecular e sua utilização para detecção de danos no DNA

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

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Bioengineering

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

11.5K visualizações.  Baylor College of Medicine. Nós demonstramos a montagem e aplicação de um dispositivo em escala molecular alimentado por uma proteína topoisomerase. A construção é um sensor de bio-molecular que rotula dois principais tipos de quebras de DNA em cortes de tecido, anexando dois fluoróforos diferentes para os seus fins.

Vídeo: In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Fabricação de Eletroquímica-DNA Biossensores para Detecção Reagentless de ácidos nucléicos, proteínas e pequenas moléculas

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Bioengenharia

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip: A alta taxa de transferência de plataforma 96-Bem, para avaliação dos danos causados ​​no DNA em células Microarrayed Humanos

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Irritação dos olhos Test (EIT) para identificação de perigos de olho irritantes químicos usando reconstruída Humano Córnea-like epitelial (RHCE) Modelo Tissue

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning e Montagem de microvasos 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Robótico líquido manipulação montagem de dispositivos modulares DNA automática

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Síntese de cationizada Magnetoferritin para a magnetização ultra-rápida de Células

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

Construção e utilização de uma câmara de estimulação elétrica para reforçar a diferenciação osteogênica em células estromais/tronco mesenquimais em Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Micromatrizes de biopartículas para análise quimiotática e molecular de swarming de nêutrons humanos in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Modelos avançados de fígado 3D para testes de genotoxicidade in vitro após exposição nanomaterial de longo prazo

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.