El estudio actual fue financiado por "Estudio sobre los problemas clave del efecto curativo de Koumiss en las enfermedades regionales de la medicina mongola" en el proyecto apoyado en 2018 del programa de ciencia y tecnología del Departamento de Ciencia y Tecnología de la Región Autónoma de Mongolia Interior.

NOTA: Consulte la Tabla de materiales para obtener detalles relacionados con todos los materiales, instrumentos y reactivos utilizados en este protocolo.
1. Cultivo celular
<ol><li>Cultivo de células HepG2 en matraces de cultivo que contienen el medio Eagle Modificado de Dulbecco (DMEM) (que contiene 10% de suero fetal bovino [FBS], 100 unidades/mL de penicilina y 100 μg/mL de estreptomicina). Mantener los matraces de cultivo a 37 °C en una incubadora con 5% de CO2 .</li>
</ol>2. Efecto del ácido oleico en la viabilidad celular medido por el kit de recuento celular-8
<ol><li>Disolver un volumen específico de OA en dimetilsulfóxido (DMSO) para lograr una concentración de 200 mM. Almacene la solución a -20 °C para su uso futuro.</li>
	<li>Siembre células HepG2 en una placa de 96 pocillos con una densidad celular de 6 × 103 celdas por pocillo. Agregue 100 μL de DMEM a cada pocillo. Incubar y cultivar las células a 37 °C en una incubadora de 5% de CO2 durante 24 h. Divida las células HepG2 en dos grupos:<ol><li>Grupo de control: añadir medio de cultivo celular.</li>
		<li>Grupo OA: añadir OA al medio de cultivo celular para alcanzar concentraciones finales de 0,125 mM, 0,25 mM, 0,5 mM y 1 mM.</li>
	</ol></li>
	<li>Después de 24 h de incubación inicial, desechar el sobrenadante de cada pocillo y agregar OA de acuerdo con el grupo especificado, agregando 100 μL por pocillo. Para el grupo de control, agregue 100 μL de medio de cultivo celular a cada pocillo. Continúe incubando las células durante otras 24 h. NOTA: Asegúrese de que cada grupo tenga seis pozos replicados. Para evitar la evaporación, agregue 100 μL de solución salina tamponada con fosfato (PBS) al anillo exterior de los pocillos en la placa de 96 pocillos.</li>
	<li>Después de 24 h de incubación, agregue 10 μL de kit de conteo de células-8 (CCK-8) a cada pocillo, mezcle suavemente e incube durante 2 h en la oscuridad. Retire la placa de 96 pocillos de la incubadora, colóquela en el lector de microplacas y mida el valor de absorbancia a 450 nm (A450). Los valores de IG50 se contabilizaron según el DO.</li>
</ol>3. Tinción de aceite rojo O para observar la formación de gotas de lípidos intracelulares
<ol><li>Siembre las células HepG2 en placas de cultivo celular de 6 pocillos a una densidad de 5 × 105 células por pocillo y cultive las placas en una incubadora de temperatura constante durante 24 h. Consulte la Figura 1 para obtener una representación visual de los pasos descritos.</li>
	<li>Después de 24 h de cultivo celular, agregue 2 mL de medio de cultivo celular que contenga OA a cada pocillo, logrando concentraciones finales de 0.125 mM, 0.25 mM, 0.5 mM y 1 mM. Después de 24 h adicionales, retire el medio de cultivo celular de cada pocillo y lave dos veces con PBS. Agregue 1 mL de fijador ORO a cada pocillo e incube durante 30 min.</li>
	<li>Prepare la solución de tinción ORO mezclando la solución de tinción A con la solución de tinción B en una proporción de 3:2. Deje reposar la mezcla a temperatura ambiente durante 10 minutos, luego filtre una vez a través de un filtro de 0,45 μm. Guarde la solución filtrada en un tubo de centrífuga protegido de la luz hasta su uso.</li>
	<li>Deseche el fijador y lávese dos veces con agua destilada. Añadir 1 mL de isopropanol al 60% a cada pocillo e incubar durante 30 s. Deseche el 60% de la solución de isopropanol y agregue 1 mL de solución de tinción ORO recién preparada a cada pocillo antes de incubar durante 20 minutos. Deseche la solución de tinción ORO, agregue 1 mL de isopropanol al 60% a cada pocillo e incube durante 30 s. Lava 5 veces con agua para eliminar el exceso de tinte.</li>
	<li>Cubra las células con agua destilada y observe bajo un microscopio. Una vez recogidas las imágenes, desecha el líquido de la placa y deja que se seque. A continuación, añada 2 mL de isopropanol a cada pocillo y agite el plato en un agitador orbital durante 10 min. Transfiera el líquido a una nueva placa de 96 pocillos, con 16 pocillos en cada grupo, agregando 100 μL por pocillo. Calcule el contenido de lípidos midiendo la densidad óptica (OD) de cada pocillo utilizando un lector de microplacas a 510 nm (A510).</li>
</ol>4. Efectos de diferentes concentraciones de ácido oleico sobre el triglicérido total en el sobrenadante celular HepG2
<ol><li>Equilibre el kit a temperatura ambiente durante 20 minutos y prepare las placas necesarias para el experimento.</li>
	<li>Recoja el sobrenadante celular y centrifugue a 1.570 × g durante 10 min. Establecer pozos estándar y probar pozos de muestra. Agregue 50 μL de concentración estándar ([S0 → S5] seguida de: 0, 0.5, 1, 2, 4, 8 mmol/L) a los pocillos estándar. Además de los pocillos en blanco y estándar, agregue 10 μL de muestras diferentes a los pocillos de muestra, seguido de agregar 40 μL de diluyente de muestra a cada pocillo. Añadir 100 μL de anticuerpo de detección-peroxidasa de rábano picante a cada pocillo, sellar con una membrana de placa e incubar a 37 °C durante 1 h en un horno de temperatura constante.</li>
	<li>Deseche el sobrenadante, séquelo en papel sin polvo y lave cada pozo con 1 solución de lavado. Dejar reposar a temperatura ambiente durante 1 min. Repita el proceso de lavado 5 veces.</li>
	<li>Agregue 50 μL de sustrato A y 50 μL de sustrato B a cada pocillo. Mezclar suavemente e incubar durante 15 min a 37 °C. Agregue 50 μL de solución de terminación a cada pocillo y mida el valor OD de cada pocillo a 450 nm (A450) dentro de los 15 min.</li>
	<li>Represente la concentración del patrón a lo largo del eje x y el valor de absorbancia (OD) correspondiente a lo largo del eje y para realizar una regresión lineal y derivar la ecuación de la curva para calcular el valor de concentración de cada muestra.</li>
</ol>5. Análisis estadístico
<ol><li>Determinar diferencias significativas en los datos cuantitativos.</li>
	<li>Calcule la media ± la desviación estándar (DE) y represente gráficamente los datos. Considere que P < 0,05 son estadísticamente significativos.</li>
</ol>

Células HepG2 inducidas por ácido oleico para el análisis de enfermedades metabólicas 

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Los autores declaran no tener conflictos de intereses.

El MASLD es un síndrome clinicopatológico caracterizado por un depósito excesivo de grasa intracelular en los hepatocitos debido a factores más allá del alcohol y otros agentes hepáticos establecidos18. La MASLD está estrechamente relacionada con la lesión hepática por estrés metabólico adquirido, especialmente asociada con la resistencia a la insulina y la susceptibilidad genética. Para estudiar y cribar eficazmente los fármacos para detectar la MASLD, es crucial seleccionar un model...

La enfermedad hepática esteatótica asociada a la disfunción metabólica (MASLD, por sus siglas en inglés) abarca una variedad de afecciones, que incluyen esteatosis simple, esteatohepatitis no alcohólica (EHNA), cirrosis y carcinoma hepatocelular 1,2,3,4,5,6, todas atribuidas a factores distintos al consumo de alcohol7. La MASLD es la enfermedad hepática más prevalente causada por lesión metabólica hepática, afectando a casi una cuarta parte de la población mundial 8,9,10,11,12. Si bien aún no se ha dilucidado la patogenia precisa de MASLD, varias teorías intentan explicar su desarrollo. Una noción predominante sugiere un alejamiento de la teoría clásica de "dos golpes" hacia un modelo de "múltiples golpes"1. Un elemento central de estas hipótesis es el papel de la resistencia a la insulina, que se cree que es fundamental en la patogénesis de MASLD13. Las investigaciones indican que la resistencia a la insulina en los hepatocitos conduce a un aumento de los niveles de ácidos grasos libres, formando posteriormente triglicéridos almacenados en el hígado14,15.
Los investigadores han utilizado modelos in vivo e in vitro para simular la deposición de grasa en MASLD; Sin embargo, replicar completamente su mecanismo patológico sigue siendo un desafío. A pesar de esta limitación, estos modelos han sido fundamentales en el estudio de posibles dianas terapéuticas para MASLD. Sin embargo, el desarrollo de un modelo estable de MASLD es crucial. Si bien los modelos animales son efectivos, requieren mucho tiempo y son costosos, lo que pone de manifiesto el creciente interés en los modelos celulares in vitro . Estos modelos suelen utilizar uno o varios ácidos grasos libres, como el ácido oleico (OA) y el ácido palmítico, para recrear el MASLD inducido por la dieta. Entre ellas, la línea celular de hepatoblastoma humano HepG2 se utiliza a menudo para establecer modelos celulares in vitro de MASLD.
La inducción de OA estimula las células HepG2 para replicar la deposición de grasa similar a MASLD, un método con una historia bien establecida. El objetivo de este estudio fue demostrar la viabilidad, la tinción de Oil Red O (ORO), el contenido de lípidos y el nivel de triglicéridos (TG) de las células HepG2 tratadas con 0,25 mM de OA. El objetivo de este experimento fue proporcionar más evidencia para el desarrollo de estudios de modelado MAFLD.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Millex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA (1x) Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;m filter</td>
			<td>Millex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Crygenic Vials</td>
			<td>CORNING</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>25 cm2 Cell Culture Flask</td>
			<td>CORNING</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plate</td>
			<td>CORNING</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well cell culture plate</td>
			<td>CORNING</td>
			<td>3599</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Count Plate</td>
			<td>Shanghai Jing Jing Biochemical Reagent &#38; Instrument Co.</td>
			<td>02270113</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Kit-8 assays</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>CA1210-1000T</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>NUAIRE</td>
			<td>NU-5710E</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;DMSO Dimethyl sulfoxide&#160;</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>D8371</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>8122691</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme Labeling Equipment</td>
			<td>Tecan</td>
			<td>Spark</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Qualified</td>
			<td>Gibco</td>
			<td>10099141</td>
			<td></td>
		</tr>
		<tr>
			<td>HepG2 cells line</td>
			<td>Beijing North China Chuanglian Biotechnology Research Institute (BNCC)</td>
			<td>221031</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Triglyceride (TG) ELISA instruction</td>
			<td>Nanjing Jiacheng Bioengineering Institute</td>
			<td>20170301</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope for Cell Culture</td>
			<td>Leica</td>
			<td>DMi1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Tianjin Zhiyuan Chemical Reagent Co.</td>
			<td>2021030141</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red Stain Kit, For Cultured Cells</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>G1262</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid&#160;</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>A502071</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS 24.0</td>
			<td></td>
			<td>Statistics software</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro modeling of fat deposition in metabolic dysfunction-associated steatotic liver disease

La prevalencia de la enfermedad hepática esteatótica asociada a la disfunción metabólica (MASLD, por sus siglas en inglés) ha aumentado debido a los cambios en los patrones económicos y de estilo de vida, lo que ha provocado importantes problemas de salud. En informes anteriores se ha estudiado el establecimiento de modelos animales y celulares para MASLD, destacando las diferencias entre ellos. En este estudio, se creó un modelo celular mediante la inducción de la acumulación de grasa en MASLD. Las células HepG2 se estimularon con el ácido graso insaturado ácido oleico a varias concentraciones (0,125 mM, 0,25 mM, 0,5 mM, 1 mM) para emular MASLD. La eficacia del modelo se evaluó mediante ensayos del kit de recuento de células-8, tinción con Oil Red O y análisis del contenido de lípidos. Este estudio tuvo como objetivo crear un modelo celular fácil de operar para las células MASLD. Los resultados de los ensayos del kit de recuento de células 8 mostraron que la supervivencia de las células HepG2 dependía de la concentración de ácido oleico, con un IG50 de 1,875 mM. La viabilidad celular en los grupos de 0,5 mM y 1 mM fue significativamente menor que en el grupo control (P < 0,05). Además, la tinción de Oil Red O y el análisis del contenido de lípidos examinaron la deposición de grasa a diferentes concentraciones de ácido oleico (0,125 mM, 0,25 mM, 0,5 mM, 1 mM) en células HepG2. El contenido de lípidos de los grupos de 0,25 mM, 0,5 mM y 1 mM fue significativamente mayor que el del grupo control (P < 0,05). Además, los niveles de triglicéridos en los grupos de OA fueron significativamente más altos que en el grupo control (P < 0,05).

Efecto del ácido oleico sobre la viabilidad celular Las células HepG2 se expusieron a concentraciones variables de OA (0 mM, 0,125 mM, 0,25 mM, 0,5 mM, 1 mM), lo que resultó en una disminución en las tasas de supervivencia celular a 0,125 mM, 0,25 mM, 0,5 mM y 1 mM en comparación con 0 mM. Se observó significación estadística a 0,5 mM (P < 0,05) y 1 mM (P < 0,05) en comparación con 0 mM. Los resultados del impacto de la OA en la viabilidad celular, evaluados por el kit CCK-8...

Autor destacado: Establecimiento de modelos celulares MASLD para investigar los mecanismos de la enfermedad y los efectos hipolipemiantes del koumiss

Este artículo describe el uso de células HepG2 inducidas por ácido oleico como modelo para la enfermedad hepática esteatótica asociada a la disfunción metabólica.

Modelado in vitro de la deposición de grasa en la enfermedad hepática esteatótica asociada a la disfunción metabólica

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading ...

in-vitro-modeling-fat-deposition-metabolic-dysfunction-associated

Research

JoVE Journal

Biology

In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-Associated Steatotic Liver Disease

412 vistas.  Inner Mongolia Medical University. Nuestra dirección de investigación es el estudio de la medicina tradicional mongola mediante el establecimiento de un modelo celular de enfermedad hepática esteatótica asociada a la disfunción metabólica. Se estudia el papel y el mecanismo de la terapia con koumiss. Los hallazgos del estudio de presencia resultarán ventajosos en el futuro, permitiendo el establecimiento de modelos celulares de enfermedad hepática esteatótica asociada a la disfunción...

Video: Autor destacado: Establecimiento de modelos celulares MASLD para investigar los mecanismos de la enfermedad y los efectos hipolipemiantes del koumiss

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself de dispositivos para la recuperación de las muestras criopreservadas de caída accidental en los tanques de almacenamiento criogénicos

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Investigación

Biología

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

El aislamiento de los ribosomas polipéptidos nacientes Bound<em&gt; In vitro</em&gt; Para la identificación de sitios de traslación pausa a lo largo del ARNm

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Usar secuencias de detención SecM como una herramienta para aislar Ribosome polipéptidos enlazados

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

En Vivo Modelado del Genoma Humano Morbid usando Danio rerio

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Metabólicas etiquetado de repeticiones ricas en leucina quinasas 1 y 2 con Radioactive Fosfato

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

La detección de las proteínas de unión a ARN por<em&gt; In Vitro</em&gt; ARN desplegable de adipocitos Cultura

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Análisis de la función E3 Ubiquitina Ligasa

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

Aislamiento de células valvulares primarias humanas para el modelado de enfermedades in vitro

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

In vitro Ensayo de germinación tridimensional de angiogénesis utilizando células madre embrionarias de ratón para el modelado de enfermedades vasculares y pruebas de fármacos

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is often not detected until drugs have reached clinical stages, risking patients' safety and leading to substantial losses for the pharma industry. Taking into account that standard 2D models have limitations in detecting DILI it is essential to develop in vitro models that are more predictive to improve data translatability. To understand causality and mechanistic aspects of DILI in detail, a human liver MPS consisting of human primary liver parenchymal and non-parenchymal cells (NPCs) and cultured in 3D microtissues on an engineered scaffold under perfusion has been developed. Cryopreserved primary human hepatocytes (PHHs) and Kupffer cells (HKCs) were cocultured as microtissues in the MPS platform for up to two weeks, and each compound of interest was repeatably dosed onto liver microtissues at seven test concentrations for up to four days. Functional liver-specific endpoints were analyzed (including clinical biomarkers such as alanine aminotransferase, ALT) to evaluate liver function. Acute and chronic exposure to compounds of various DILI severities can be assessed by comparing responses to single and multi-dosed microtissues. The methodology has been validated with a broad set of severe and mildly hepatotoxic compounds. Here we show the data for pioglitazone and troglitazone, well-known hepatotoxic compounds withdrawn from the market for causing liver failures. Overall, it has been shown that the liver MPS model can be a useful tool to assess DILI and its association with changes in hepatic function. The model can additionally be used to assess how novel compounds behave in distinct patient subsets and how toxicity profiles may be affected by liver disease states (e.g., viral hepatitis, fatty liver disease).

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

Drug-induced liver injury (DILI) is a major cause of drug failure. A protocol has been developed to accurately predict the DILI liability of a compound using a liver microphysiological system (MPS). The liver model uses the coculture of primary hepatic cells and translationally relevant endpoints to assess cellular responses to treatment.

Sistema microfisiológico hepático humano para evaluar la toxicidad hepática inducida por fármacos in vitro

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is ...