Os autores declararam que não há conflito de interesses.

O protocolo global requer 2 dias, incluindo o primeiro dia da indução de sepse e isolamento do fluido BAL de animais e o segundo dia para isolamento de exosomes do mouse BALF. Todos os procedimentos foram revistos e aprovados pelo Comitê de uso no centro médico do VA de Atlanta e institucional Cuidado Animal.
1. modelo de lesão pulmonar aguda séptico Mouse
<ol><li>Use 6-8 semanas - velho selvagem-tipo C57BL/6J camundongos machos (20 a 22 g de peso) para o animal modelo e executar todos os procedimentos utilizando técnicas assépticas e instrumentos. Autoclave todos os instrumentos cirúrgicos, incluindo fórceps e a tesoura por 20 min a 121 ° C.</li>
	<li>Distribua aleatoriamente os ratos na experimental e grupo controle. Controle de ratos em um quadro de cirurgias. Trate ratos com Escherichia coli lipopolissacarídeo (LPS, 15 mg/kg) por via intraperitoneal. De acordo com o peso do corpo individual, diluir dosagens adequadas de LPS de 0,1 mL de PBS por rato, em seguida, injetar solução LPS utilizando uma seringa de 1 mL com agulha 27G e injetar uma quantidade equivalente de PBS para controle de animais.</li>
	<li>Manter os ratos na instalação de BSL2 animal, que consiste em gaiolas limpas à temperatura adequada.</li>
	<li>24h depois, eutanásia em ratos com inalação de CO2 .</li>
	<li>Selecione a área cirúrgica no centro do pescoço do mouse. Faça uma pequena incisão (cerca de 5 mm) e separar suavemente os músculos para o acesso à traqueia usando fórceps rombudo até a traqueia é exposta.</li>
	<li>Retire o fluido de BAL rato com seringa estéril de 10 mL com agulha 27G. Delicadamente, introduza a agulha na traqueia e injectar 1 mL de PBS estéril lentamente para a traqueia, depois desenhar BALF uma seringa descartável de 3 mL com agulha 27G. Após a conclusão do procedimento, descarte de seringas e agulhas em recipiente de objectos cortantes de risco biológico.</li>
	<li>Tratar de instrumentos cirúrgicos com dióxido de cloro, conforme descrito anteriormente por Chauret et al 27e em seguida esterilizar todos os instrumentos cirúrgicos em autoclave por 20 min a 121 ° C.</li>
</ol>2. Exosomes isolamento-série Ultracentrifugação e caracterização
<ol><li>Recolha o líquido de BAL rato nos tubos cônico de 15 mL. A colheita de células alveolares, centrifugar amostras durante 10 minutos a 1.000 g, a 4 ° C.</li>
	<li>Transferir o sobrenadante para novos tubos de 15ml cónico e, em seguida, centrifugar amostras durante 30 min a 4.000 x g a 4 ° C para remover os resíduos de célula.</li>
	<li>Recolha o sobrenadante em tubos se e centrifugar amostras para 60 min a 10.000 x g a 4 ° C para remover as partículas maiores do celulares.</li>
	<li>Transferir o sobrenadante para novos tubos cónicos e, em seguida, filtrar as amostras através de 0,2 µm seringa-filtro para remover restantes partículas maiores.</li>
	<li>Coletar amostras em tubos de se e equilibrar os tubos. Centrifugar as amostras por 2 h a 140.000 x g a 4° C para o exosomes de Pelotas. Nota: O tamanho de exosomes varia de 20-200 nm. 0,2 µm filtro é usado para a remoção de partículas maiores que são mais de 200 nm de tamanho.</li>
	<li>Descarte os sobrenadantes suavemente. Dissolva a pelota com 100 µ l PBS ou solução de Lise e transferência exosomes amostras para tubos de 1,5 mL e armazenar a-80 ° C até o uso. Nota: Microscópio eletrônico de transmissão é recomendado para verificar exosomes isolado.</li>
	<li>Siga a amostra-padrão TEM protocolo28de processamento. Analisar amostras de exosomes usando microscopia eletrônica de transmissão e tirar fotos, escala bar 200 nm.</li>
</ol>3. preparação de miRNA e Performing miRNA-q-RT-PCR
Nota: miRNA-q-RT-PCR é aconselhável analisar o nível de expressão dos miRNAs.
<ol><li>Seguir o protocolo de isolamento padrão miRNAs e certifique-se de usar uma quantidade igual de RNAs purificadas na transcrição reversa do único-fio24.</li>
	<li>Siga o protocolo padrão de PCR em tempo real para detectar o nível de microRNA exosomal no fluido de BAL. Uso específicos iniciadores de PCR em tempo real contra miR-155 e miR-146 U6 SnRNA. Executar a reação de PCR com o aparelho PCR quantitativo e normalizar os valores de expressão para controle interno U6 snRNA. Nota: Cada grupo consiste de três vias amostras24.</li>
</ol>4. Exosomes de rotulagem e de purificação
<ol><li>Suspender o sedimento exosomes com solução de suspensão de 100 µ l em um tubo de polipropileno de 1,5 mL e, em seguida, adicione 0,4 µ l do corante exosomes no outro tubo com 100 µ l da solução de suspensão.</li>
	<li>Rapidamente adicionar exosomes na solução para a solução de corante e misture completamente por pipetagem. Manter amostras por 5 min à temperatura ambiente, proteger da luz.</li>
	<li>Colocar uma coluna de rotação exossomo em um tubo de eluição de 1,5 mL. Transfira a mistura de solução para o centro da coluna exossomo de rotação.</li>
	<li>Gire a coluna por 2 min a 800 x g, à temperatura ambiente.</li>
	<li>Descartar a coluna e armazenar a amostra eluted a-20 ° C até o uso.</li>
</ol>5. validação de Exosomes Up tomadas pelo destinatários células
<ol><li>Sementes de 1 x 104 12 MLE /cell em um slide de câmara de 8 poços e células de cultura a 37 ° C, 5% de CO2.</li>
	<li>Adicione 10 µ l fluorescente-etiquetadas exosomes para as células. Incubar durante 1 h a 37 ° C.</li>
	<li>Remover os meios de cultura e lavam-se células com PBS e, em seguida, corrigir células com paraformaldeído 1% por 10 minutos em temperatura ambiente.</li>
	<li>Monte o slide no meio de montagem com DAPI.</li>
	<li>Analisar as amostras através de um microscópio de fluorescência e tirar fotos, ampliação de 40 X.</li>
</ol>6. verificação da interação entre Exosomes e células destinatários
<ol><li>Semente de 2 x 105 MLE12/poço em uma placa de cultura de 12 poços, contendo meio de cultura apropriado e células de cultura a 37 ° C, 5% de CO2.</li>
	<li>Adicionar 20 µ g exosomes de fluido do mouse BAL para células MLE12 e configurar repetições por grupo experimental.</li>
	<li>24h depois, lavam-se células com PBS e adicionar tampão de Lise 600 µ l em poço. Homogeneizar a placa e coletar amostras de Lise no tubo de 1,5 mL com pipeta.</li>
	<li>Siga o protocolo de isolamento de RNA total padrão e protocolo de transcrição reversa do cDNA, completar a transcrição reversa de cDNA da único-Costa29.</li>
	<li>Siga o protocolo padrão de PCR em tempo real para detectar o nível de expressão do mRNA em células destinatário29. Uso específicos iniciadores de PCR em tempo real contra o TNF-α, IL-6 e ZO-1. Executar as reações de PCR com o aparelho PCR quantitativo e normalizar os valores de expressão para o controle interno GAPDH. Nota: Cada grupo consiste de amostras de três vias.</li>
</ol>

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Este trabalho foi apoiado pela revisão de mérito VA 2I01BX001786-06A1 financiamento para RS.

Modelos de mouse de doenças são comumente usados para avaliar a função fisiológica de genes específicos e para reduzir o custo de experimentação2. A lesão pulmonar aguda séptico descrita aqui imita a resposta inflamatória nos seres humanos com SDRA. Este modelo é relevante para investigar a patogénese molecular, desenvolvimento de biomarcadores e testar potenciais novas terapias5.
Sistemas de co-cultura são relevantes para estudos <...

ALI e Sara é as formas de vida-ameaçando de insuficiência respiratória com hipoxemia grave causada por edema pulmonar não-cardiogênico, que afeta cerca de 1 milhão pessoas no mundo anualmente1. A etiologia da SDRA inclui lesão direta para os pulmões de infecções ou aspiração e uma variedade de insultos indiretas. Na última década, tem havido uma maior compreensão da patogênese molecular da SDRA, no entanto, tratamentos alvo específicos para SDRA estão ainda a ser desenvolvido,2,3.
Foram desenvolvidos vários modelos animais de lesão pulmonar aguda que fornecem uma ponte para traduzir terapias experimentais para estudos humanos4,5. Modelos comumente usados incluem a instalação local do ácido oleico, bactérias, LPS e bleomicina. Outras abordagens incluem isquémia-reperfusão, punção cecal ligadura, lesão de estiramento induzida pela ventilação mecânica, hiperóxia ou administração sistémica de bactérias e LPS-5. Esses modelos fornecem um sistema biológico útil para testar hipóteses clínicas e para o desenvolvimento de terapias potenciais. Para simular a SDRA humana, modelos animais devem reproduzir inflamação e lesão aguda para as células epiteliais e endoteliais com defeitos na função de barreira nos pulmões.
Exosomes são vesículas de membrana com 20-200 nm de diâmetro, cujo conteúdo molecular contém proteínas, DNA, RNA e lipídios, e facilitar a comunicação intercelular no microambiente tecido via transferência de composição molecular. Exosomes são secretadas por vários tipos de células, como células endoteliais, células epiteliais, células musculares lisas e células tumorais e existem em fluidos do corpo humano. Estudos indicam que a exosomes regular conversas cruzadas entre células do estroma e células do sistema imunológico durante doenças inflamatórias, infecciosas e estéril, e sua liberação anormal parece ser regulada por vários estímulos naturais e experimentais durante fisiológica e processos patológicos6. Essa rede de comunicação pode desempenhar um importante papel na patogênese de doenças pulmonares e pode afetar a progressão fisiopatológicos7,8. 18-22 como nucleotídeos RNAs não-codificantes, miRNAs existem em ambos os tecidos e fluidos corporais, plasma, soro e modulam a expressão de RNAm no nível pós-traducional9,10.
MiRNAs embalados em exosomes influenciam a diferenciação e função dos vários tipos de células, e níveis excessivos são associados com uma variedade de doenças, incluindo câncer, doenças pulmonares, obesidade, diabetes e doença cardiovascular11, 12,13,14,15,16. Entrada em células da destinatários e vaivém de miRNAs exosomal facilitar a comunicação intercelular, modificando a hemostasia do microambiente17,18. Lesão pulmonar aguda é um processos complexos, envolvendo vários tipos de células com extensa comunicação intercelular através de exosomes8. miR-155 e miR-146 compartilham o mecanismo regulador transcricional comum e contribuam para a resposta inflamatória e a tolerância imunológica19,20. Estudos recentes indicam que ambos modulam a resposta inflamatória via exosomal miRNAs vaivém entre células do sistema imunológico21. No entanto, os mecanismos moleculares subjacentes moduladora efeitos de miRNAs exosomal na resposta alveolar a endotoxina permanecem pouco claras, sem dúvida, o potencial relevância clínica e translacional implicação mérito mais investigações.
Modelos de co-cultura estão sendo empregados para definir a interação dos tipos de célula específica no complexo ambiente, tais como inflamação e câncer22,23. Estas plataformas fornecem uma estratégia alternativa para interrogar conversas cruzadas entre tipos de células, particularmente por células imunes e estruturais.
Intratraqueal, aerossol, intraperitoneal ou sistêmica administração de LPS são amplamente utilizados para induzir a lesão de pulmão experimental a24,25,26e mostraram para induzir permeabilidade endotelial e epitelial defeitos de fabricação. Aqui usamos LPS intraperitoneal para induzir um fossa séptico modelo de lesão pulmonar aguda em camundongos. Dentro de 24 horas da administração intraperitoneal de LPS, permeabilidade defeitos são induzidos nos pulmões com recrutamento de células inflamatórias. Além disso, mostramos que exosomes de BAL contêm miRNA-155 e miR-146, e exosomes de fluido BAL induzir a expressão de citocinas pró-inflamatórias em pilhas epithelial do destinatários, incluindo IL-6 e TNF-α. Estes dados são os primeiros a mostrar que os miRNAs exosomal são secretadas no BAL neste modelo de lesão pulmonar aguda séptico.

<table border="0" cellpadding="0" cellspacing="0" style="width:1276px;" width="1276">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:691px;">lipopolysaccharide</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:224px;">Escherichia coli 055:B5</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS pH7.2(1X)</td>
			<td>Life technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mirVana miRNA isolation kit</td>
			<td>Thermo Fisher</td>
			<td align="right">449774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>Thermo Fisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mmu-miR-155 (002571) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">has-miR-146a (000468) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U6snRNA primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TNF-&#945;(Mm00443258_m1 ) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IL-6 (Mm00446190_m1&#160;&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ZO-1 (Mm00493699_m1&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minimal essential medium (MEM)</td>
			<td>Thermo Fisher</td>
			<td>11095-072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trysin-EDTA solution(0.05%)</td>
			<td>Thermo Fisher</td>
			<td align="right">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosomes were labeled with PKH67 through PKH67 Green Fluorescent Cell linker Mini Kit&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>MINI67-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosome Spin Columns (MW3000)</td>
			<td>Thermo Fisher</td>
			<td align="right">4484449</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Beckman Coulter Optima L-100XP ultracentrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td>L-100XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-clear centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td align="right">344058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Applied Biosystems 7500 Fast Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">transmission electron microscopes (TEM)</td>
			<td>JEOL Ltd</td>
			<td>120 kV TEM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olympus BX41 microscope</td>
			<td>Olympus</td>
			<td>BX41</td>
			<td></td>
		</tr>
	</tbody>
</table>

bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury

Lesão pulmonar aguda (LPA) e síndrome da angústia respiratória (Sara) representam um grupo heterogêneo de doenças pulmonares, que continua a ter uma alta taxa de morbidade e mortalidade. A patogénese molecular de ALI está sendo melhor definido; no entanto, devido à natureza complexa da doença terapias moleculares ainda precisam ser desenvolvido. Aqui nós usamos um modelo de rato induzida de lipopolissacarídeo (LPS) de lesão pulmonar aguda séptico para delinear o papel do exosomes na resposta inflamatória. Usando este modelo, fomos capazes de mostrar que os ratos expostos a LPS intraperitoneal secretam exosomes no líquido de lavagem (BAL) bronco-alveolar dos pulmões que são embalados com miRNA e citocinas que regulam a resposta inflamatória. Ainda mais usando um sistema de modelo co-cultura, mostramos que exosomes lançados de macrófagos interromper a expressão de proteínas de junção apertada nas células epiteliais brônquicas. Estes resultados sugerem que 1) conversas cruzadas entre inatas células imunes e estruturais através do vaivém exosomal contribuem para a resposta inflamatória e rompimento de barreira estrutural e 2) segmentação destes miRNAs pode fornecer uma nova plataforma para tratar ALI e SDRA.

Para induzir lesão pulmonar séptico, os ratos foram tratados com LPS intraperitoneal (15 mg/kg). Dentro de 24 horas da administração de LPS, neutrofílico influxo foi visto nos pulmões, conforme mostrado na figura 1A. Fluido de mouse BAL foi desenhado, seguido pelo isolamento e purificação de exosomes. Morfologia do LBA exosomes foi confirmada por microscopia eletrônica de transmissão (figura 1B).
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Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

Exosomes de lavagem broncoalveolar em lesão pulmonar séptico induzidas pelo lipopolissacarídeo

Os ratos expostos ao LPS intraperitoneal secretam exosomes no líquido de lavagem (BAL) bronco-alveolar que são embalados com miRNAs. Usando um sistema de co-cultura, mostramos que exosomes lançados no fluido BAL perturbará a expressão de proteínas de junção apertada nas células epiteliais brônquicas e aumentar a expressão de citocinas pró-inflamatórias que acentuam a lesão pulmonar.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

bronchoalveolar-lavage-exosomes-lipopolysaccharide-induced-septic

Research

JoVE Journal

Genetics

10.8K visualizações.  Atlanta VAMC. Os ratos expostos ao LPS intraperitoneal secretam exosomes no líquido de lavagem (BAL) bronco-alveolar que são embalados com miRNAs. Usando um sistema de co-cultura, mostramos que exosomes lançados no fluido BAL perturbará a expressão de proteínas de junção apertada nas células epiteliais brônquicas e aumentar a expressão de citocinas pró-inflamatórias que acentuam a lesão pulmonar.

Vídeo: Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

As análises siRNA transfecção e EMSA sobre recentemente isolados das vilosidades citotrofoblastos Humanos

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Genética

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

In Situ Rotulagem de replicação de DNA mitocondrial em Drosophila Adulto Ovários por Edu Coloração

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

A mutagénese aleatória optogenetic Usando Histona-miniSOG em<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

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

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

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

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

Criação e Double-stranded mediada por RNA silenciamento de genes na Esconder Beetle,<em&gt; Dermestes maculatus</em

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

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

Em pool shRNA tela para reativação de MeCP2 no cromossomo X inativo

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

Formaldeído-assistida isolamento de elementos reguladores para acessibilidade de cromatina medida em células de mamíferos

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Mediada por CRISPR reorganização da estrutura de Loop de cromatina

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Identificação de eventos de recombinação homóloga em células-tronco embrionárias de camundongo usando Southern Blotting e reação em cadeia da polimerase

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Caracterizando a histona modificação pós-traducional alterações nos modelos de Neurodegenerative Proteinopathy de levedura

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

Determinação da taxa de fertilização do ovo de Bemisia tabaci usando uma técnica citogenética

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...