Agradecemos ao Prof. Weiguo Zou e à S. Kato pelos reagentes e ratos e aos membros do laboratório Zou por discussões úteis. Agradecemos também ao Laboratório de Estomatologia Digitalizada e Centro de Pesquisa de Anomalias Craniofaciais do Hospital do Nono Povo de Xangai pela assistência. Este trabalho foi apoiado em parte por subvenções da Fundação Nacional de Ciência Natural da China (NSFC) [81570950,81870740,81800949], Shanghai Summit & Plateau Disciplines, o fundo SHIPM-mu do Instituto de Medicina de Precisão de Xangai, Shanghai Nona Escola Popular de Medicina, Shanghai Jiao Tong University School of Medicine [JC201809], o Projeto de Incentivo da Equipe de Inovação de Alto Nível para a Escola de Medicina da Universidade de Xangai Jiao Tong , o Fundo de Pesquisa Interdisciplinar do Hospital do Nono Povo de Xangai, Shanghai JiaoTong University School of Medicine [JYJC201902]. E L.J. é um estudioso dos Talentos Médicos Da Juventude, Shanghai "Rising Stars of Medical Talent" Youth Development Program e o projeto "Chen Xing" da Universidade de Xangai Jiaotong.

Todos os métodos relacionados aos animais aqui descritos foram aprovados pelo Comitê Institucional de Cuidados e Uso de Animais (IACUC) da Faculdade de Medicina da Universidade de Xangai Jiaotong.
1. Reprodução de camundongos de exclusão Stat3 específicos do assino
NOTA: Os ratos Stat3fl/fl foram obtidos comercialmente. Os camundongos Ctsk-Cre foram fornecidos pela S. Kato (Universidade de Tóquio, Tóquio, Japão12). Os camundongos foram criados e mantidos sob condições específicas sem patógenos (FPS) na instalação institucional de animais em condições padronizadas.
<ol><li>Emparelhe um rato macho sexualmente maduro com duas fêmeas da mesma idade. Após 18 dias, verifique diariamente os cheques para recém-nascidos. Separe os camundongos fêmeas grávidas e mantenha-o sozinho, se necessário. Mude os camundongos machos entre diferentes gaiolas de reprodução se os camundongos fêmeas não estivessem grávidas dentro de um mês do emparelhamento.</li>
	<li>Cruze ratosfl/fl de Stat3para ratos Ctsk-Cre (F0). Corte as caudas para genotipagem e mantenha o Stat3fl/+ ;masculino Camundongos Ctsk-Cre até que estejam sexualmente maduros, que tem cerca de 6 semanas de idade (F1). Use os seguintes primers: Stat3 F-TTGACCTGTGCTCCTACAAAAAAAAAAA; Stat3 R-CCCTAGATTAGGCCAGCACA; Ctsk-cre F-GAACGCACTGATTTCGACCA; Ctsk-cre R-GCTAACCAGCGTTTTCGTTC.</li>
	<li>Cruze 6 semanas de idade masculino Stat3fl/+; Camundongos Ctsk-Cre com ratos Stat3fl/fl femininos. Corte as caudas para genotipagem e mantenha o Stat3 fl/flmasculino; Camundongos Ctsk-Cre até que estejam sexualmente maduros, que tem cerca de 6 semanas de idade (F2).</li>
	<li>Cruz 6-week-old masculino Stat3 fl/fl; Camundongos Ctsk-Cre com ratos Stat3fl/fl fêmeas (F3). Corte as caudas para genotipagem e substitua os camundongos de criação antigos por camundongos mais jovens com o tempo (F3+N).</li>
</ol>2. Coleta de espécimes
<ol><li>Eutanize seis pares de 8 semanas de idade Stat3fl/fl e Stat3Ctsk lixo-mate ratos separadamente com asfixia por dióxido de carbono. NOTA: A taxa de fluxo de CO2 desloca 30% do volume da gaiola por minuto (por exemplo, para 45 cm x 30 cm x 30 cm de gaiola a taxa de fluxo de CO2 é de 40 L/min).</li>
	<li>Coloque os ratos em uma posição supina. Desloque as articulações bilaterais do quadril suavemente à mão. Use uma tesoura oftálmica para cortar a pele verticalmente da tíbia distal e, em seguida, dissecar toda a pele do membro traseiro.</li>
	<li>Corte o ligamento articular da articulação do quadril direito e articulação do joelho com uma tesoura para separar o membro traseiro. Corte o trochanter e a junção da fíbula e, em seguida, mergulhe o membro traseiro em 4% de paraformaldeído. Mantenha os membros traseiros certos para o passo 3. Corte adequadamente o osso em ambas as extremidades para imergir completamente e fixar a medula óssea com 4% de paraformaldeído.</li>
	<li>Corte o ligamento articular da articulação do quadril esquerdo e da articulação do joelho com uma tesoura, remova suavemente o tecido mole e separe cuidadosamente a tíbia e o fêmur. Mergulhe a tíbia e o fêmur separadamente em 75% de etanol. Mantenha a femora para a etapa 4 e tíbia para a etapa 6. Certifique-se de manter o trochanter intacto.</li>
</ol>3. Preparação da seção de parafina
<ol><li>Fixar o membro traseiro direito em 4% de paraformaldeído a 4 °C por 48 h.</li>
	<li>Decalcificar: Lave suavemente os espécimes com 1x PBS por 10 min 3 vezes. Decalcifice os espécimes em 15% EDTA (150 g EDTA em 800 mL de ddH2O e 100 mL de 10x PBS) com um decalcificador ultrassônico por 3 a 4 semanas até que os ossos possam ser dobrados. Substitua por um fluido de decalcificante fresco a cada dois dias.</li>
	<li>Lave suavemente as amostras 3x com 1x PBS e, em seguida, mergulhe-as em 75% de etanol a 4 °C durante a noite.</li>
	<li>Desidratação: No segundo dia, imergir sequencialmente espécimes em 95% de etanol, 100% etanol e xileno, cada um por 1h duas vezes.</li>
	<li>Mergulhe espécimes em 1/2 xileno 1/2 parafina por 30 min. Mergulhe os espécimes em parafina a 65 °C durante a noite.</li>
	<li>Incorporar: Selecione um tanque de incorporação adequado para incorporar. Coloque a tíbia uniformemente por baixo. Coloque o fêmur e a tíbia em um ângulo de 90°. Depois que a parafina tiver esfriado e solidificado, remova-a do tanque de incorporação. Numerar os espécimes e armazená-los a -20 °C durante a noite.</li>
	<li>Corte seções de 5 μm de espessura continuamente usando o microtoma. Corte de 20 a 40 seções. Espalhe as seções em água de 37 °C, adere-as aos slides do microscópio e asse a 42 °C durante a noite.</li>
</ol>4. Micro-CT de digitalização e análise
<ol><li>Escaneie a femora esquerda com um scanner microcáludo. Resolução: 10 μm; Tensão: 70 kV; Corrente: 114 μA; Fliter: 0,5 mm Al; Passo de rotação: 0,5°.</li>
	<li>Reconstrua imagens 3D do osso cortical e do osso trabecular usando o software de suporte do scanner seguindo as instruções do fabricante. Os ROIs têm uma largura total de 1 mm de osso trabecular próximo à placa de crescimento distal e em uma seção total de 1 mm de largura do osso cortical no meio da femora.</li>
	<li>Calcular os parâmetros quantitativos de microarquitetura: densidade mineral óssea (DMO), fração de volume ósseo (VB/TV), espessura trabecular (Tb.Th.), número trabecular (Tb.N.), separação trabecular (Tb.Sp.) e espessura óssea cortical (Ct.Th.).</li>
</ol>5. Coloração de armadilha
<ol><li>Asse as seções de parafina a 65 °C por 30 min.</li>
	<li>Dewax: Mergulhe as seções em xileno por 10 minutos. Realize esta etapa 3x com xileno fresco.</li>
	<li>Reidratar: Mergulhe as seções sequencialmente em 100% etanol, 95% etanol, 70% etanol e ddH2O, cada uma por 5 min duas vezes.</li>
	<li>Prepare a solução de coloração usando o kit de coloração TRAP seguindo as instruções do fabricante e aqueça até 37 °C. NOTA: A solução de coloração TRAP deve ser preparada imediatamente antes de cada ensaio.</li>
	<li>Adicione 50-100 μL de solução de coloração a cada amostra e incubar em uma câmara úmida de 37 °C por 20-30 min. Verifique o estado de coloração dos osteoclastos sob um microscópio de luz a cada 5 minutos até que os osteoclastos multinucleados vermelhos possam ser vistos. Termine a reação com ddH2O.</li>
	<li>Contra-mancha na solução de hematoxilina para 30 s. Crie uma cor azul estável por imersão em solução de amônia de 1% por 1 min. Enxágüe lentamente água da torneira.</li>
	<li>Monte as seções com tampas usando bálsamo neutro e seque durante a noite.</li>
	<li>Capture 3-5 campos de interesse por um microscópio. Analise o perímetro trabecular por Imagem J: meça o comprimento da barra de escala (Ls) usando a ferramenta 'linha reta' como L1, em seguida, meça o comprimento do perímetro trabecular usando a ferramenta 'linha segmentada' como L2, o comprimento físico (Lp)= Ls*L2 /L1). Conte o número de células TRAP-positivas com mais de três núcleos.</li>
</ol>6. Rotulagem dupla vermelha de Calcein e alizarin
<ol><li>Preparação da amostra: Injete intraperitonealmente 20 mg/kg de calceína (1 mg/mL em solução NaHCO3) no dia 0, e 25 mg/kg alizarin vermelho S (AL, 2 mg/mL em H2O) no dia 4. Sacrifício de ratos no 7º dia. Desassociar cuidadosamente a tíbia e fixar em 4% paraformaldeído a 4 °C por 48 h.</li>
	<li>Desidratar: Após a fixação, lave suavemente a tíbia 3x com 1x PBS. Imersão sequencialmente as amostras em 95% de etanol, 100% etanol e xileno por 5 min duas vezes separadamente.</li>
	<li>Mergulhe os espécimes em acetona por 12 h, em 1/2 resina acetona 1/2 por 2 h, e em resina pura em um forno de secagem durante a noite. NOTA: A resina foi preparada por resina comercialmente disponível (por exemplo, Embed 812 Resin) de acordo com as instruções do fabricante.</li>
	<li>Incorporar: Adicione resina pura em um tanque adequado de incorporação de gel de sílica e coloque os espécimes suavemente para evitar bolhas. Polimerize a resina em forno de secagem a 60 °C por 48 h.</li>
	<li>Corte os espécimes em seções de 5 μm de espessura continuamente com um microtome rotativo. Armazene o resto das amostras com dessecante à temperatura ambiente.</li>
	<li>Adere às seções com pinças em uma queda de 75% de álcool. Monte as seções com tampas usando bálsamo neutro. Capture rotulagem de fluorescência vermelha e verde com um microscópio de fluorescência.</li>
	<li>Meça a largura entre duas linhas de rotulagem (Ir.L.Wi), perímetro trabecular de etiqueta única (sL.Pm), perímetro trabecular de dupla rotulagem (dL.Pm) e perímetro trabecular total (Tb.Pm). Calcular a taxa de apposition mineral (MAR) e a taxa de formação óssea (BFR/BS). MAR = Ir.L.Wi/dias de intervalo. BFR/BS= (dL.Pm±sL.Pm/2)*MAR/Tb.Pm*100 %.</li>
</ol>

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Modelos de camundongos geneticamente modificados são comumente usados para estudar o mecanismo e o tratamento farmacêutico da doença humana13. Os camundongos Ctsk-Cre têm sido amplamente utilizados para estudos funcionais de osteoclatos6. O presente estudo descreveu os protocolos dos métodos para analisar o fenótipo esquelético e estudar os genes críticos que controlam a atividade osteoclasta in vivo.
A análise histológica é o...

O osso esquelético é o principal órgão portador de carga do corpo humano e está sob pressão do ambiente interno e externo durante a caminhada e o exercício1. Ao longo de sua vida, os ossos passam continuamente pela auto-renovação, que é equilibrada por osteoblastos e osteoclartos. O processo de osteoclasstos limpando ossos velhos e osteoblastos formando novos ossos mantém a homeostase e a função mecânica do sistema esquelético2. A perturbação no equilíbrio pode induzir doenças metabólicas ósseas, como a osteoporose. A osteoporose, causada pelo excesso de atividade osteoclástica, é globalmente prevalente e causa perdas econômicas substanciais para a sociedade2,3,4. De acordo com o número limitado de medicamentos disponíveis para o tratamento da osteoporose e seu risco de efeitos adversos4, é importante desvendar os detalhes da formação e atividade do osteoclato.
Os osteoclatos derivados da linhagem hematopoiética monócito/macrófago têm múltiplos núcleos (podem ter de 2 a 50 núcleos) e são grandes (geralmente maiores que 100 μm de diâmetro)2. Embora a exploração de mecanismos e a triagem de medicamentos para distúrbios osteoclásticos tenham sido amplamente melhoradas através da cultura osteoclasta in vitro, as complicadas reações orgânicas tornam a evidência in vivo indispensável para a terapia-alvo. Devido às semelhanças genéticas e fisiopacionológicas entre camundongos e humanos, modelos de camundongos geneticamente modificados são comumente utilizados para estudar os mecanismos e os tratamentos farmacêuticos da doença humana in vivo6. O sistema Cre-loxP é uma tecnologia amplamente utilizada para edição de genes de camundongos e permitiu que os pesquisadores investigassem funções genéticas de forma específica de tecido/célula5. Cathepsin K (CSTK) é uma protease de cisteína secretada por osteoclartos que pode degradar colágeno ósseo8. É bem aceito que o CTSK seja expresso seletivamente em osteoclastos maduros; portanto, os camundongos Ctsk-Cre são considerados uma ferramenta útil para estudos funcionais de osteoclatos e tem sido utilizado6.
O transdutor de sinal e ativador da família de transcrição (STAT) é clássico e altamente significativo em imunidade e progressão e desenvolvimento do câncer7,8. Entre os sete STATs, o STAT3 é relatado como o mais relevante para a homeostaseóssea 9,10. Vários estudos in vivo relataram que a inativação específica do STAT3 nos osteoblastos diminui a formaçãoóssea 9,10. No entanto, as evidências sólidas sobre a participação do STAT3 na formação de osteoclatos e metabolismo ósseo in vivo ainda são limitadas. Recentemente, fornecemos evidências in vivo com um modelo de mouse de exclusão Stat3 específico do osteoclasta(Stat3fl/fl; Ctsk-Cre, a partir de agora chamado Stat3Ctsk) que o STAT3 participa da diferenciação osteoclasta e do metabolismo ósseo11. No presente estudo, descrevemos os métodos e protocolos que utilizamos para analisar as alterações na massa óssea, histomorfologia óssea e anabolismo ósseo e catabolismo dos camundongos Stat3Ctsk, a fim de estudar a influência da exclusão STAT3 específica do osteoclato na homeostase óssea.

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	<tbody>
		<tr>
			<td>4% Paraformaldehyde solution</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>E672002</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>80000360</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonia solution</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Ctsk-Cre mice</td>
			<td></td>
			<td></td>
			<td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td>
		</tr>
		<tr>
			<td>DDSA</td>
			<td>Electron Microscopy Sciences</td>
			<td>13710</td>
			<td></td>
		</tr>
		<tr>
			<td>DeCa RapidlyDecalcifier</td>
			<td>Pro-Cure</td>
			<td>DX1100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP-30</td>
			<td>Electron Microscopy Sciences</td>
			<td>13600</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>EMBED 812 RESIN</td>
			<td>Electron Microscopy Sciences</td>
			<td>14900</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescence microscope</td>
			<td>Olympus</td>
			<td>IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin solution</td>
			<td>Beyotime Biotechanology</td>
			<td>C0107</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-CT</td>
			<td>Scanco Medical AG</td>
			<td>&#956;CT 80</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>10018918</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>E675007</td>
			<td></td>
		</tr>
		<tr>
			<td>NMA</td>
			<td>Electron Microscopy Sciences</td>
			<td>19000</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>A601889</td>
			<td></td>
		</tr>
		<tr>
			<td>rotary microtome</td>
			<td>Leica</td>
			<td>RM2265</td>
			<td></td>
		</tr>
		<tr>
			<td>Stat3fl/fl mice</td>
			<td>GemPharmatech Co., Ltd</td>
			<td>D000527</td>
			<td></td>
		</tr>
		<tr>
			<td>TRAP staining kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

skeletal phenotype analysis of a conditional stat3 deletion mouse model

Modelos de camundongos transgênicos são poderosos para entender os genes críticos que controlam a diferenciação e atividade osteoclante, e para estudar mecanismos e tratamentos farmacêuticos da osteoporose. Os camundongos Cathepsin K (Ctsk)-Cre têm sido amplamente utilizados para estudos funcionais de osteoclatos. O transdutor de sinal e ativador da transcrição 3 (STAT3) é relevante na homeostase óssea, mas seu papel nos osteoclas in vivo permanece mal definido. Para fornecer a evidência in vivo de que o STAT3 participa da diferenciação osteoclasta e do metabolismo ósseo, geramos um modelo de rato de exclusão Stat3 específico do osteoclasta(Stat3 fl/fl; Ctsk-Cre) e analisou seu fenótipo esquelético. A varredura microcsi tomografia e a reconstrução 3D implicaram aumento da massa óssea nos camundongos eliminatórios condicionais. Foram realizadas colorações de H&E, re coloração dupla de calceína e alizarina vermelha e coloração de fosfattase de ácido resistente a tartaratos (TRAP) para detectar o metabolismo ósseo. Em suma, este protocolo descreve alguns métodos e técnicas canônicas para analisar o fenótipo esquelético e estudar os genes críticos que controlam a atividade osteoclasta in vivo.

Utilizando o protocolo atual, foram gerados camundongos de exclusão Stat3 específicos para estudar a influência da exclusão de STAT3 na diferenciação de osteoclatos. Os camundongos Stat3Ctsk e seus ninhados wildtype (WT) foram criados e mantidos após a genotipagem. Macrófagos de medula óssea foram isolados e cultivados em osteoclatos, e a exclusão de STAT3 em camundongos Stat3Ctsk foi demonstrada(Figura 1).
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Watch this Scientific Journal Video about Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model at JoVE.com

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Este protocolo descreve um método canônico para entender os genes críticos que controlam a atividade osteoclasta in vivo. Este método utiliza um modelo de camundongo transgênico e algumas técnicas canônicas para analisar o fenótipo esquelético.

Análise do fenótipo esquelético de um modelo de rato de exclusão stat3 condicional

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

skeletal-phenotype-analysis-conditional-stat3-deletion-mouse

Research

JoVE Journal

Biology

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

4.3K visualizações.  National Clinical Research center of Stomatology. Este protocolo descreve um método canônico para entender os genes críticos que controlam a atividade osteoclasta in vivo. Este método utiliza um modelo de camundongo transgênico e algumas técnicas canônicas para analisar o fenótipo esquelético.

Vídeo: Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biologia

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Análise do desenvolvimento de um fenótipo morfológico como uma função da concentração de proteínas em levedura de brotamento

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo de imagens e tratamentos terapêuticos em um modelo de camundongo ortotópico de câncer de ovário

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Culturas alternativas para pluripotentes estaminais humanas produção de células, Manutenção e Análise Genética

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Detecção e análise de danos no DNA em Rato Músculo Esquelético<em&gt; In Situ</em&gt; Usando o método TUNEL

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

Cryosectioning da contíguos Regiões de um músculo esquelético único mouse para expressão do gene e análises histológicas

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-interno pilha injeção maciça de células-tronco embrionárias leva a taxas mais elevadas de quimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Análise de espectrometria de massa para identificar a ubiquização do CENP-A marcado pelo EYFP (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Isolamento das Mitocôndrias do Músculo Esquelético do Rato para Ensaios Respirométricos

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...