Funding from the Kansas Agricultural Experiment Station and K-State Arthropod Genomics Center to KYZ supported the original development of chitosan/dsRNA-mediated gene silencing in A. gambiae19.The A. gambiae work described here was supported in part by R01-AI095842 from NIH/NIAID to KM. Development of chitosan/siRNA-mediated gene silencing in A. aegypti20,21 was supported by NIH/NIAID Award R01-AI081795 to MDS. The contents of this study are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

1. Mosquito Especies y Crianza <ol><li> Mantener A. gambiae G3 y A. cepas aegypti Liverpool IB12 (utilizados en los estudios representativos abajo) u otras cepas de interés de acuerdo con la práctica de laboratorio estándar o como se describe previamente 23,24. </li></ol> 2. dsRNA y siRNA Diseño y Producción <ol><li> Diseño cebadores para construir plantillas de ARN de doble cadena largos específicos para el gen de interés. Utilice la herramienta de E-RNAi 25. Producir dsRNA de acuerdo con el método de elección o como se describió previamente 19. <ol><li> Para un control negativo, sintetizar dsRNA 19 correspondiente a la secuencia de la proteína fluorescente verde (GFP), β-galactosidasa (β-gal), o algún otro gen no se expresa en los mosquitos. Si así se desea, dsRNA 19 utilizado para generar los resultados representativos se resumen a continuación se puede utilizar como un control positivo. Tienda dsRNA disuelve en agua RNasa libre a -80 °; C hasta que se necesite. </li></ol></li><li> Por otra parte, el diseño de genes específicos de siRNA de acuerdo con la práctica estándar de laboratorio o como se describe anteriormente 10. Mezcle la secuencia de un siRNA desmontables para diseñar ARNsi de control negativo que no corresponde a cualquier gen de mosquitos. Compra los siRNAs personalizados, que están disponibles a través de un número de proveedores de confianza. <ol><li> Si así se desea, siRNAs 20,21 utilizado para obtener los resultados representativos se resumen a continuación se puede utilizar como controles positivos. Tienda siRNA disuelve en agua libre de ARNasa a -80 ° C hasta que se necesite. </li></ol></li></ol> 3. Preparación Chitosan / ARN de interferencia nanopartículas <ol><li> Reúna sulfato de sodio RT 100 mM pre-hechos (100 mM Na 2 SO 4 en desionizada H 2 O) y acetato de sodio 0,1 M (0,1 M NaCl 2 H 3 O 2 -0,1 M de ácido acético, pH 4,5 en desionizada H 2 O) tampones. </li><li> Disolver quitosano (≥75%desacetilada) en tampón de acetato de sodio 0,1 M para hacer 0,02% (w / v) de solución de trabajo y mantener la solución a temperatura ambiente antes de su uso. </li><li> Disolver dsRNA o siRNA en 50 l H 2 O desionizada y añadirlo al tampón de sulfato de sodio 100 mM para hacer una solución de 100 l de 32 g de dsRNA / siRNA por 100 l de sulfato de sodio 50 mM. </li><li> Añadir 100 l de solución de quitosano a la solución de dsRNA / siRNA y luego calentar la mezcla en un baño de agua a 55 ° C durante 1 min. Establecer un control mediante la adición de 100 l de sulfato de sodio 50 mM a 100 l de solución de quitosano y seguir el mismo procedimiento. </li><li> Mezclar las soluciones inmediatamente durante 30 segundos mediante agitación de alta velocidad a temperatura ambiente para facilitar la formación de nanopartículas. </li><li> Centrifugar la mezcla a 13.000 xg durante 10 min a TA, después de lo cual un pellet debe ser visible. Transferir el sobrenadante a un nuevo tubo de 1,5 ml. El aire seco de la pastilla para ≈10 min a RT antes de usarla para preparar la comida de los mosquitos. </li><li> MEDIDA la concentración de dsRNA / siRNA en el sobrenadante utilizando el sobrenadante de la control (ver 3.5) como un blanco para calcular la cantidad total de dsRNA / siRNA que permaneció en el sobrenadante. Utilice la diferencia entre las cantidades de partida de dsRNA / siRNA y las cantidades restantes en los sobrenadantes para calcular el porcentaje de dsRNA / siRNA atrapado en las nanopartículas. Esta eficiencia de carga es normalmente más del 90%. </li><li> Repita el mismo procedimiento si se necesitan más nanopartículas. Utilice las nanopartículas secas inmediatamente. El impacto de almacenamiento en frío de partículas antes de su uso no ha sido evaluada. </li></ol> 4. Preparación Mosquito Alimentos que contienen quitosano / ARN de interferencia Nanopartículas <ol><li> Prepare 1 ml de una solución de agarosa al 2% (w / v) en agua desionizada, fundir la agarosa, y mantener la solución de agarosa fundida en un baño de 55 ° C el agua antes de su uso. </li><li> Mezclar alimentos en escamas de pescado (47% de proteína cruda, min. 10% de grasa cruda, max. De fibra cruda 3%) y levadura seca en unarelación de 2: 1 (w / w). Triturar la mezcla de pequeñas partículas con un mortero (transitable a través Nº 50 EE.UU. tamiz de prueba estándar). Utilice la comida suelo, que debe ser de color marrón, ya sea inmediatamente o almacenarlo en un contenedor sellado a 4 ° C durante varias semanas. </li><li> En un tubo de 1,5 ml, mezclar 6 mg de alimentos suelo con las nanopartículas secas de la Sección 3.7 con un palillo. </li><li> Añadir 30 l de solución de gel de agarosa al 2% pre-fundida a la mezcla de nanopartículas de alimentos; revuelva inmediatamente ya fondo mediante el uso de una punta de un palillo o una pipeta. </li><li> Utilice el gel que contiene alimentos y nanopartículas para alimentar a las larvas de mosquitos inmediatamente. Alternativamente, una vez que el gel se solidificó completamente a TA, almacenar el gel a 4 ° C y utilizar el día siguiente, o a -80 ° C para su uso posterior. </li></ol> 5. La alimentación de larvas de mosquitos con alimentos que contengan quitosano / ARN de interferencia Nanopartículas <ol><li> Alimentación A. gambiae larvas de mosquitos: <ol><li> Retirar un SIpellet gel ngle del tubo de 1,5 ml con un palillo y lo dividirá en 6 rebanadas iguales utilizando una hoja de afeitar limpia o palillo de dientes. </li><li> Transferencia de 20 larvas de tercer instar a un plato de Petri de 500 ml que contiene 100 ml de agua desionizada. </li><li> Alimentar a las larvas de mosquito mediante la adición de una rebanada de la pastilla de gel (finamente picado en trozos más pequeños) por placa de Petri de una vez al día durante cuatro días. Asegúrese de observar las larvas se alimentan de la pastilla, que debería ser significativamente reducida en tamaño o completamente ausentes por el día siguiente. Después de un tiempo, los mosquitos se convertirán en larvas de cuarto instar tarde. </li><li> Grabe los cambios fenotípicos visibles durante el experimento. Examinar los niveles de transcripción y otros cambios fenotípicos como se discutió en la sección 6 al final del período de cuatro días. </li></ol></li><li> Alimentación A. aegypti larvas: <ol><li> Cortar el pellet gel en 6 rebanadas iguales utilizando una hoja de afeitar limpia o un palillo. </li><li> Coloque 50-sincronizado edad de 24 horas después de la eclosión del huevo filarvas de instar primero en una placa de Petri en ≈40 ml de agua desionizada. </li><li> Las larvas se alimentan un corte por placa de Petri durante 4 horas / día, a continuación, transferir las larvas de nuevo a la dieta de las larvas regular de 2: 1 de tierra, alimentos en escamas de pescado y levadura seca para el resto del día. Repita el procedimiento diario a través de los cuatro estadios larvales. </li><li> Examinar los niveles de transcripción y otros cambios fenotípicos como se discutió en la sección 6 en los puntos de tiempo de desarrollo deseados. </li></ol></li></ol> 6. Confirmación del gen Desmontables <ol><li> Relativo cuantificación transcripción de QRT-PCR en A. aegypti y A. gambiae larvas. <ol><li> Realizar y analizar los ensayos de qRT-PCR con tres réplicas biológicas en al menos 10 de control agrupada vs. larvas experimental como se describe 11,19,20, o de acuerdo con el procedimiento estándar de laboratorio. Los resultados representativos se incluyen a continuación. </li><li> Para el análisis de una parte particular del tipo de tejido / cuerpo (es decir., El cerebro o antena), perform QRT-PCR como se describe en 6.1.1 después de la disección para recuperar el tejido de interés (por ejemplo, ver Mysore et al. 21). </li></ol></li><li> Confirmación de caída por hibridación in situ <ol><li> Sintetizar antisentido digoxigenina-etiquetados y sentir ribosondas de control de acuerdo con la práctica estándar de laboratorio o como se describe 26. </li><li> Ejecutar la hibridación in situ por el protocolo Haugen et al. 27 para la confirmación de caída como se describió anteriormente 11,20 y discutido en la sección de resultados representativos a continuación. </li></ol></li><li> Confirmación de caída por inmunohistoquímica <ol><li> Si se dispone de anticuerpos contra el producto proteico del gen objetivo, realizar inmunohistoquímica como se ha descrito previamente 28, que facilita la identificación de los individuos con los mayores niveles de caída para el análisis de fenotipo 20 como se ejemplifica en la representative resultados sección de abajo. </li></ol></li></ol>

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The authors have nothing to disclose.

El / interferir metodología de nanopartículas de quitosano ARN descrito en este documento se ha utilizado para dirigir eficazmente genes durante el desarrollo larval en A. gambiae (Figuras 2, 3) y A. aegypti (Figuras 4, 5, 6, los cuadros 1, 2). Nanopartículas de quitosano pueden ser preparadas con cualquiera de largo dsRNA o siRNA, ambos de los cuales se han utilizado con éxito en los mosquitos como se evidencia por los resultados representativos descritos en el pr...

Los mosquitos se alimentan de sangre de vectores de los géneros anofelinos y Aedine transmiten agentes patógenos responsables de varios de los peores flagelos de la humanidad. Se estima que unos 3,4 mil millones de personas están en riesgo de contraer la malaria, que es responsable de más de medio millón de muertes al año en todo el mundo. Resultados de la malaria de la infección por Plasmodium sp. Parásitos, que se transmiten al ser humano por la picadura de mosquitos infectados del género Anopheles, incluyendo el vector africano director Anopheles gambiae ( <a href="http://www.who.int/topics/malaria/en/">http://www.who.int/topics/malaria/en/</a> , 2014) 1. Aedes aegypti es el mosquito vector primario para el virus del dengue, que causa la fiebre del dengue, una enfermedad febril inespecífica que es la enfermedad por arbovirus más extendida e importante en el mundo. El virus del dengue es actualmente una amenaza a&gt; 2,5 mil millones de personas en los trópicos, con una inciden anualce de aproximadamente 50 millones de casos con resultado de ~ 24.000 muertes al año ( <a href="http://www.cdc.gov/dengue/">http://www.cdc.gov/dengue/</a> , 2014) 2. A pesar del impacto mundial devastadora de enfermedades transmitidas por mosquitos en la salud humana, medio eficaz de prevención y tratamiento de estas enfermedades son escasas. El control de mosquitos es actualmente el mejor método de prevención de enfermedades. El potencial para el control de enfermedades transmitidas por artrópodos por la manipulación genética de los insectos vectores ha sido reconocido por más de cuatro décadas 3. Cepas transgénicas de A. aegypti diseñados para tener un volador fenotipo femenino específico represible han hecho recientemente la posibilidad de utilizar las estrategias de control de vectores transgénicos una realidad 4-6. Estos avances han desafiado a los investigadores a identificar dianas genéticas novedosas para el control de vectores y otros medios de manipulación de genes en función de los mosquitos vectores. La alteración de la expresión génica durante el desarrollo, como fue el caso en las mujeres no volador mosquitos 4, puede promover el esclarecimiento de las estrategias de control de vectores novedosos. Sin embargo, en gran parte debido a las dificultades técnicas, las funciones de muy pocos genes se han caracterizado durante el desarrollo de A. gambiae, A. aegypti, o de otras especies de mosquitos. Desde su descubrimiento en C. elegans 7, RNA de interferencia (RNAi), que se conserva en los animales, plantas y microorganismos, se ha utilizado ampliamente para estudios genéticos funcionales en una amplia variedad de organismos, incluyendo insectos 8,9. La vía de RNAi se inicia por Dicer, que escinde largo dsRNA en 20-25 siRNAs cortos-nucleótidos de largo que funcionan como ARN de interferencia específico de secuencia. genes silencio siRNAs que son complementarias en la secuencia mediante la promoción de rotación de transcripción, escote, y la interrupción de la traducción 9. Largas moléculas de dsRNA (típicamente 300-600 pb) o siRNAs personalizadas dirigidas a una particulasecuencia r se puede utilizar en el laboratorio de investigación para silenciar cualquier gen de interés. Mediante la gestión de ARN de interferencia cuando se entrega, los investigadores pueden controlar el tiempo en el que el silenciamiento de genes iniciados. Esta ventaja es útil, ya que se puede utilizar para superar retos como la letalidad del desarrollo o la esterilidad, lo que puede dificultar la producción y mantenimiento de cepas con mutaciones hereditarias, un proceso costoso y laborioso que aún no está disponible en todas las especies de insectos. Aunque el grado de silenciamiento génico por RNAi puede variar de un gen a gen, tejido a tejido, y un animal a otro, RNAi es ampliamente utilizado para el análisis funcional de los genes en los mosquitos y otros insectos 8,9. Tres estrategias de entrega de interferencia de ARN se han utilizado en los mosquitos: la microinyección, la aplicación de remojo / tópica, y la ingestión. Para una historia detallada y comparación del uso de estas tres técnicas en los insectos, consulte Yu et al 8. We han utilizado con éxito microinyección 10 como un medio de entrega de siRNAs para dirigir genes de desarrollo en A. aegypti embriones, larvas y pupas 11-14. Sin embargo, esta estrategia de prestación de mano de obra intensiva requiere tanto una configuración de microinyección y una mano experta. Por otra parte, la microinyección es estresante para el organismo, un factor de confusión, en particular cuando se evaluarán fenotipos conductuales. Por último, la entrega microinyección no puede extenderse al campo para el control de vectores. Como alternativa, empapando el organismo en interferir solución de ARN también se ha convertido en un medio popular para inducir el silenciamiento de genes, ya que es conveniente y requiere poco equipo o el trabajo. El remojo principalmente se ha aplicado en la línea celular de insecto estudia 8, pero en un estudio reciente, desmontables se logró en A. aegypti sumerge en una solución de dsRNA 15, que los animales parecían estar ingerir. Sin embargo, para los estudios con análisis de múltiples Experimentagrupos l o fenotipos, remojo es bastante costoso. López-Martínez et al. 16 describe la rehidratación impulsado RNAi, un nuevo enfoque para la entrega de ARN de interferencia que implica la deshidratación en solución salina y la rehidratación con una sola gota de agua que contiene ARN de interferencia. Este enfoque no corta los costos asociados con la inmersión de animal completo, pero es más caro que la microinyección y puede ser limitada en su aplicación a las especies que pueden tolerar altas presiones osmóticas. Por otra parte, es difícil imaginar cómo la inmersión o la metodología de inmersión / rehidratación deshidratación podrían ser adaptados para el control de vectores en el campo. Por estas razones, para estudios post-embrionarias, la entrega de ARN de interferencia con los alimentos ingeridos es una estrategia alternativa viable. Aunque las estrategias basadas en la ingestión no funcionan en todas las especies de insectos, tal vez sobre todo Drosophila melanogaster, la administración oral de ARN de interferencia se mezcla con los alimentos ha promovido si genlencing en una variedad de insectos 8,17, incluyendo A. aegypti adultos 18. Describimos quitosano nanopartículas mediada RNAi en A. larvas gambiae 19 y se han aplicado con éxito este enfoque para la reducción de la expresión de genes en A. Aegypti 20,21. Aquí, la metodología para este procedimiento RNAi, que implica el atrapamiento del ARN de interferencia por el quitosano polímero, es detallada. Chitosan / interferencia de ARN nanopartículas están formadas por autoensamblaje de policationes con ARN de interferencia a través de las fuerzas electrostáticas entre las cargas positivas de los grupos amino en el quitosán y las cargas negativas llevadas por los grupos fosfato en la cadena principal de ARN de interferencia 19. El procedimiento descrito es compatible con las moléculas de ARN de doble cadena larga (en adelante como dsRNA) o doble cadena siRNA (en lo sucesivo, siRNA). Después de la síntesis, quitosano / interferencia nanopartículas de ARN se mezclan con el alimento de las larvas y se entregan alarvas a través de la ingestión oral. Esta metodología es relativamente barato, requiere poco equipo y mano de obra 19, y facilita el análisis de alto rendimiento de múltiples fenotipos, incluyendo el análisis de los comportamientos de 20,21. Esta metodología, que puede ser adaptado para estudios de silenciamiento de genes en otros insectos, incluyendo otros vectores de enfermedades y plagas de insectos agrícolas, lo que potencialmente podría ser utilizado para el silenciamiento de genes en una variedad de otras especies animales. Además, el quitosano, un polímero barato, no tóxico y biodegradable 22, lo que potencialmente podría ser utilizado en el campo para el control de mosquitos específico de la especie.

<table border="0" cellpadding="0" cellspacing="0" width="1429">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Name of Reagent/Equipment</td>
			<td style="width:243px;">Company</td>
			<td style="width:177px;">Catalog Number</td>
			<td style="width:663px;">Comments/Descriptions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.02 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR20</td>
			<td>for resuspension of interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR200</td>
			<td>for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5 ml graduated tube&#160;</td>
			<td>Fischer Scientific</td>
			<td>05-408-120</td>
			<td>for aliquoting resuspended interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR1000</td>
			<td>for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 ml graduated tube&#160;</td>
			<td>Fischer Scientific</td>
			<td>05-408-046</td>
			<td>for preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1M Sodium Acetate, pH 4.5</td>
			<td>TEKnova</td>
			<td>S0299</td>
			<td>to prepare sodium acetate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic Acid, AIRSTAR. ACS, USP, FCC Grade</td>
			<td>BDH</td>
			<td>VWR BDH3092-500MLP</td>
			<td>to prepare sodium acetate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose Genetic Technology Grade</td>
			<td>MP Biomedicals LLC.</td>
			<td>800668</td>
			<td>to coat prepared interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Eppendorf AG</td>
			<td>5415D</td>
			<td>to pellet interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chitosan, from shrimp shells</td>
			<td>Sigma-Aldrich</td>
			<td>C3646-25G</td>
			<td>to combine with interfering RNA for prepararation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dry Yeast</td>
			<td>Universal Food Corp</td>
			<td>NA</td>
			<td>to prepare mosquito larval food with interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E-RNAi tool</td>
			<td>German Cancer Research Center</td>
			<td>NA</td>
			<td>for design of dsRNA; http://www.dkfz.de/signaling/e-rnai3//</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">goldfish food</td>
			<td>Wardley</td>
			<td>Goldfish FLAKE FOOD</td>
			<td>to prepare mosquito larval food with interfering RNA/nanoparticles</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Heated water bath</td>
			<td>Thermo Scientific</td>
			<td>51221048</td>
			<td>to heat the interfering RNA/nanoparticles at 55oC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice</td>
			<td>not applicable</td>
			<td>not applicable</td>
			<td>for thawing interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice bucket</td>
			<td>Fisher Scientific</td>
			<td>02-591-44</td>
			<td>for storage of ice used during the procedure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">liver powder</td>
			<td>MP Biomedicals LLC.</td>
			<td>900396</td>
			<td>to feed mosquito larvae post interfering RNA/nanoparticle treatment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microwave oven</td>
			<td>A variety of vendors</td>
			<td>not applicable</td>
			<td>to prepare 2% agarose solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">petridish (100X15 mm)</td>
			<td>Fischer Scientific</td>
			<td>875713</td>
			<td>interfering RNA/nanoparticle feeding chamber for larvae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH meter</td>
			<td>Mettler Toledo</td>
			<td>S220</td>
			<td>for preparation of buffers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Razor blade</td>
			<td>Fischer Scientific</td>
			<td>12-640</td>
			<td>to divide the interfering RNA/nanoparticle pellet for feedings</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">siRNA</td>
			<td>Thermo Scientific/Dharmacon</td>
			<td>custom</td>
			<td>for preparation of siRNA/nanoparticles</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sodium Sulfate, Anhydrous (Na2SO4)</td>
			<td>BDH/distributed by VWR</td>
			<td>VWR BDH0302-500G</td>
			<td>to prepare 50mM sodium sulfate solution in which the interfering RNA will be resuspended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermometer</td>
			<td>VWR</td>
			<td>61066-046</td>
			<td>to measure the water bath temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tooth picks</td>
			<td>VWR</td>
			<td>470146-908</td>
			<td>for stirring during interfering RNA/nanoparticle food preparation and cutting gel pellets</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ultralow freezer</td>
			<td>A variety of vendors</td>
			<td>not applicable</td>
			<td>for storage of interfering RNA aliquots and interfering RNA/nanoparticles at -80oC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortex mixer</td>
			<td>Fischer Scientific</td>
			<td>02-216-108</td>
			<td>for preparation of chitosan/interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Weight paper</td>
			<td>Fischer Scientific</td>
			<td>NC9798735</td>
			<td>to divide the interfering RNA/nanoparticle pellet for feedings</td>
		</tr>
	</tbody>
</table>

chitosan/interfering rna nanoparticle mediated gene silencing in disease vector mosquito larvae

Mosquitos vectores infligen más sufrimiento humano que cualquier otro organismo - y matan a más de un millón de personas cada año. Los proyectos del genoma del mosquito facilitaron la investigación en nuevas facetas de la biología de los mosquitos, incluidos estudios genéticos funcionales en el principal vector de la malaria Anopheles gambiae africana y el dengue y la fiebre amarilla vector Aedes aegypti. ARN de interferencias (RNAi-) mediada por el silenciamiento de genes se ha utilizado para dirigir genes de interés en ambas de estas especies de mosquitos vectores de enfermedades. Aquí se describe un procedimiento para la preparación de quitosano / interferir nanopartículas de ARN que se combinan con los alimentos y ingeridos por las larvas. Este técnicamente sencilla, de alto rendimiento, y la metodología relativamente barato, que es compatible con una larga ARN de doble cadena (dsRNA) o pequeños ARN de interferencia (siRNA) moléculas, se ha utilizado para la caída con éxito de un número de diferentes genes en A. gambiae y A. aegyptilarvas. Siguiendo la alimentación de larvas, desmontables, que se verifica a través de QRT-PCR o hibridación in situ, pueden persistir por lo menos a través de la fase de pupa tarde. Esta metodología puede ser aplicable a una amplia variedad de mosquitos y otras especies de insectos, incluyendo las plagas agrícolas, así como otros organismos no modelo. Además de su utilidad en el laboratorio de investigación, en el futuro, quitosano, un polímero barato, no tóxico y biodegradable, podría potencialmente ser utilizada en el campo.

<html> A. gambiae: Nanopartículas de quitosano / dsRNA se forman debido a la interacción electrostática entre los grupos amino de quitosano y los grupos fosfato de dsRNA. La eficiencia de la incorporación de dsRNA en nanopartículas es usualmente por encima de 90%, medido por el agotamiento de dsRNA de la solución. Imágenes de microscopía de fuerza atómica muestran que el quitosano-dsRNA promedios de tamaño de partícula 140 nm de diámetro, que van desde 10...

Watch this Scientific Journal Video about Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae at JoVE.com

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Aquí se describe un procedimiento para inhibir la función génica en los mosquitos vectores de enfermedades mediante el uso de quitosano / interferir nanopartículas de ARN que son ingeridas por las larvas.

El quitosano / ARN de interferencia mediada por nanopartículas silenciamiento génico en la enfermedad vectorial larvas de mosquitos

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

chitosaninterfering-rna-nanoparticle-mediated-gene-silencing-disease

Research

JoVE Journal

Biology

14.2K vistas.  Kansas State University.  Aquí se describe un procedimiento para inhibir la función génica en los mosquitos vectores de enfermedades mediante el uso de quitosano / interferir nanopartículas de ARN que son ingeridas por las larvas. 

Video: Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Inducida por el virus de silenciamiento génico (VIGS) en Nicotiana benthamiana Y tomate

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Investigación

Biología

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Imágenes en vivo del calcio celular combinado con el silenciamiento de genes mediada por siRNA Identifica Ca 2 + Canales de fugas en la membrana del ER y sus mecanismos de regulación

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Mediada por Agrobacterium inducida por el virus Gene ensayo silenciamiento en algodón

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

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 ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

El agotamiento de ARN ribosomal de metagenómica intestino del mosquito RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

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, ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Análisis de RNA Global Síntesis en el nivel de células individuales siguiente Hipoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Evaluación del huso meiótico en ovocitos de ratón por silenciamiento siRNA mediada

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Disección y tinción de gotas de lípidos de los ovenocitos en larvas de Drosophila

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...