Este trabajo fue financiado en parte por el NIH R01 # DE021475 (CTM), una subvención del NIH predoctoral de Formación 5T32GM007127 (PAE), y una beca de investigación de Graduados de la NSF (NAE). Damos las gracias a Kevin Schwalbach para realizar recombinería y las inyecciones de BAC, Nick Donde para la generación de datos de secuenciación de Sanger CRISPR, y Katherine Lipari de información útil sobre el protocolo de inyección.

Los autores no tienen nada que revelar.

La inyección de embriones de espinosos de una sola célula para la transgénesis o la edición genoma presenta tres retos principales. En primer lugar, en relación con embriones de pez cebra, el pez espinoso embrionaria corion es difícil y, a menudo romper las agujas. Este problema puede superarse parcialmente mediante el uso de micropipetas de vidrio más gruesas y más fuertes y la inyección perpendicular al corion (véase el Protocolo, la Figura 2). Asegurar que tan poco agua como sea posible se ...

Un componente fundamental de la comprensión de cómo surge la biodiversidad es la determinación de las bases genéticas y de desarrollo de los cambios fenotípicos evolucionado en la naturaleza. El pez espinoso de tres espinas, Gasterosteus aculeatus, se ha revelado como un excelente modelo para estudiar la base genética de la evolución. Espinosos han sufrido muchos cambios evolutivos de adaptación como los peces marinos han colonizado un sinnúmero de ambientes de agua dulce en todo el hemisferio norte, lo que resulta en morfológica dramática, fisiológicos y cambios de conducta 1. Los genomas de individuos de veintiún poblaciones de espinosos se han secuenciado y ensamblado, y un mapa de ligamiento de alta densidad se ha generado para mejorar aún más el conjunto de 2,3. Experimentos de mapeo genéticos han identificado regiones genómicas subyacente fenotipos evolucionado 4-6, y en unos pocos casos, el papel funcional de genes candidatos específicos se han probado 7,8. Un número de regiones genómicas que generan cambios morfológicos se han identificado con prometedores genes candidatos, pero estos candidatos no han sido aún probado funcionalmente 9 - 12 El. Además, los espinosos son modelos comunes para estudios de genética de poblaciones / genómica 13,14, 15 especiación, comportamiento, endocrinología 1 16 17, ecotoxicología, la inmunología y parasitología 18 19. Los estudios futuros en cada uno de estos campos se beneficiarán de la capacidad de realizar manipulaciones genéticas funcionales en los espinosos. Además de la manipulación de sus secuencias de codificación, las funciones de los genes candidatos pueden evaluarse mediante el estudio de sus secuencias cis-regulador y aumentando funcionalmente, disminuyendo o eliminando la expresión del gen candidato. Métodos de microinyección y la transgénesis en los espinosos están bien establecidos 7,8,20 y fueron inicialmente desarrollados usando una meganucleasa mediada21 método descrito por primera vez en 22 medaka. El método de microinyección modificado que aquí se presenta ha sido optimizada tanto para la transgénesis mediada por Tol2 y recientemente desarrollado reactivos de edición del genoma incluyendo Talens y CRISPRs. Se cree que los cambios en los elementos cis-regulador a ser crítica para la evolución morfológica, como cis-regulador cambios pueden evitar las consecuencias negativas de las mutaciones pleiotrópicos 23 de codificación. Por lo tanto, las pruebas y la comparación de secuencias cis-regulador putativo se ha convertido en un objetivo central de un número creciente de estudios evolutivos. Además, la mayoría de las variantes de la enfermedad humanos son variantes de regulación 24,25 y sistemas modelo de vertebrados son muy necesarios para estudiar cis-regulador Función del elemento y la lógica. Los peces que fertilizar sus embriones externamente en un gran número de vertebrados ofrecen sistemas potentes para estudiar -Reglamento cis. El sistema de transposón Tol2, en el que foreign ADN de integrarse en el genoma está flanqueado por Tol2 transposasa sitios de unión y co-inyectados con ARNm transposasa Tol2, funciona con alta eficiencia para la integración con éxito plásmido construye en los genomas de peces 26 - 28. Típicamente, un promotor de potencial se clona aguas arriba de un promotor basal (como hsp70l 29) y el gen reportero fluorescente tal como EGFP (proteína fluorescente verde potenciada) o mCherry en una cadena principal de Tol2 y se inyecta con transposasa mRNA 26. La observación de la expresión del indicador fluorescente, ya sea en embriones o descendientes con transgenes integrados de forma estable inyectados, proporciona información acerca de la regulación espacial y temporal de la expresión génica impulsado por el potenciador putativo. En experimentos adicionales, potenciadores validados se pueden utilizar para conducir la sobreexpresión específica de tejido de genes de interés. Para el análisis de las regiones cis-regulador más grandes, de alta calidad genoma a gran insertobibliotecas IC mediante cromosomas artificiales bacterianos (BAC) se han construido tanto para los peces espinosos marinos y de agua dulce 30. Estos BAC se pueden recombineered para reemplazar un gen con un gen indicador fluorescente en el contexto de un grande (150 a 200 kb) región genómica 31. El indicador fluorescente se expresa entonces en un patrón espacio-temporal según lo determinado por las secuencias reguladoras dentro de la CAV. Para los estudios en los peces, los sitios Tol2 se pueden añadir a la BAC para facilitar la integración genómica 32,33. En etapas posteriores del desarrollo cuando la hibridación in situ es técnicamente difícil, la lectura fluorescente de la BAC se puede utilizar para estudiar los patrones de expresión de genes, como se ha demostrado para la proteína morfogenética ósea 6 stickleback (BMP6) 20. Además, los patrones de expresión fluorescentes en un individuo pueden ser rastreados a través del tiempo, que no se puede lograr con la hibridación in situ. BAC también se puede utilizar para añadir un additional copia de una región genómica para aumentar la dosis de un gen de interés. Para el estudio de la función génica, edición genoma es un campo en expansión explosiva que se puede utilizar para producir cambios dirigidos a secuencias genómicas en una amplia variedad de organismos 34. Transcripción nucleasas efectoras del tipo activador de (Talens) son nucleasas modulares, específicos de secuencia originalmente aisladas a partir de patógenos de plantas que pueden ser diseñados precisamente para unirse directamente a una secuencia genómica de elección y generar una doble hebra romper 35,36. Agrupados espaciadas regularmente repeticiones palindrómicas cortas (CRISPR) / sistemas CAS fueron encontrados originalmente en bacterias y usan una guía de ARN y la proteína Cas9 para generar una ruptura en una secuencia de ADN diana complementaria a la guía 37. La posterior reparación de la rotura de doble cadena creada por ambos Talens y CRISPRs menudo deja tras de sí una pequeña inserción o deleción, lo que puede alterar la función de la secuencia diana35-37. En los espinosos, Talens se han utilizado para alterar la expresión de genes por la orientación un promotor de 20, y ambos Talens y CRISPRs han producido con éxito mutaciones en las secuencias (datos no publicados) de codificación. Un protocolo detallado para la generación de CRISPRs para uso en el pez cebra se puede utilizar como una guía para desarrollar CRISPRs para los espinosos 38. Transgénicos y genoma experimentos de edición requieren introducción de ácidos nucleicos en un embrión de una célula recién fertilizado. Mediante la introducción de la herramienta de transgén o de edición de genoma temprano en el desarrollo, se maximiza el número de células hijas genéticamente manipulados en el embrión. embriones inyectados se criban entonces visualmente para fluorescencia o molecularmente examinados para modificaciones del genoma. Si las células que contribuyen a la línea germinal están dirigidos con éxito, el transgén o mutación se pueden transmitir a un subconjunto de la descendencia, incluso cuando la letalidad post-inyección es alta. Los peces pueden ser de mosaico o outcrossedentrecruzan y su descendencia tamiza para recuperar los alelos mutantes o un transgén integrado de forma estable de interés. Este protocolo describe métodos para introducir los transgenes y reactivos de edición del genoma en embriones de espinosos de una sola célula y el seguimiento de las modificaciones que darán éxito.

<table border="0" cellpadding="0" cellspacing="0" width="647">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Stereomicroscope with transillumination</td>
			<td style="width:145px;">Leica</td>
			<td style="width:119px;">S6e/ KL300 LED</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Manual micromanipulator</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">MM33</td>
			<td style="width:167px;">Marzhauser M33 Micromanipulator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pressure Injecion system</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">MPPI-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Back pressure unit</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">BPU</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette holder kit</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">MPIP</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnetic base holder</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">Magnetic base</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Foot switch</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">FSW</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iron plate (magnetic base)</td>
			<td style="width:145px;">Narishige</td>
			<td style="width:119px;">IP</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flaming/Brown Micropipette Puller</td>
			<td style="width:145px;">Sutter Instrument</td>
			<td style="width:119px;">P-97</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable transfer pipettes</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:119px;">13-711-7M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5% phenol red in DPBS</td>
			<td style="width:145px;">Sigma&#160;</td>
			<td style="width:119px;">P0290</td>
			<td style="width:167px;">injection tracer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">#5 forceps, biologie dumoxel&#160;</td>
			<td style="width:145px;">Fine Science Tools</td>
			<td style="width:119px;">11252-30</td>
			<td style="width:167px;">for needle breaking</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette Storage Jar</td>
			<td style="width:145px;">World Precision Instruments</td>
			<td style="width:119px;">E210</td>
			<td>holds needles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">6&#34;, 6 teeth per inch plaster drywall saw</td>
			<td style="width:145px;">Lenox</td>
			<td>20571 (S636RP)</td>
			<td>hold eggs for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">13 cm x 13 cm glass plate</td>
			<td style="width:145px;">any hardware store</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Borosilicate glass capillaries, 1.0 mm OD/0.58 mm ID&#160;</td>
			<td style="width:145px;">World Precision Instruments</td>
			<td style="width:119px;">1B100-F4</td>
			<td style="width:167px;">*harder glass than zebrafish injection capillaries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">150 x 15mm petri dish</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:119px;">FB0875714</td>
			<td>raise stickleback embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 x 10mm petri dish</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:119px;">08-757-100A</td>
			<td>store eggs pre-injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Instant Ocean Salt</td>
			<td style="width:145px;">Instant Ocean</td>
			<td>SS15-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Bicarbonate</td>
			<td style="width:145px;">Sigma</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricaine methanesulfonate/MS-222</td>
			<td style="width:145px;">Western Chemical Inc</td>
			<td style="width:119px;">MS222</td>
			<td style="width:167px;">fish anaesthesia/euthanasia</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sp6 transcription kit</td>
			<td style="width:145px;">Ambion</td>
			<td style="width:119px;">AM1340</td>
			<td style="width:167px;">For transcription of TALENs and transposase mRNA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RNeasy cleanup kit</td>
			<td style="width:145px;">Qiagen</td>
			<td style="width:119px;">74104</td>
			<td style="width:167px;">purify transposase or TALEN RNA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">QiaQuick PCR cleanup kit</td>
			<td style="width:145px;">Qiagen</td>
			<td style="width:119px;">28104</td>
			<td style="width:167px;">clean up plasmids for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K 20 mg/ml</td>
			<td style="width:145px;">Ambion</td>
			<td style="width:119px;">AM2546</td>
			<td style="width:167px;">for DNA preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nucleobond BAC 100 kit</td>
			<td style="width:145px;">Clontech</td>
			<td style="width:119px;">740579</td>
			<td>for BAC DNA preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NotI</td>
			<td style="width:145px;">NEB</td>
			<td style="width:119px;">R0189L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phusion polymerase</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:119px;">F-530L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qiagen PlasmidPlus Midi kit</td>
			<td style="width:145px;">Qiagen</td>
			<td style="width:119px;">12943</td>
			<td style="width:167px;">contains endotoxin rinse buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">QIAQuick Gel Extraction</td>
			<td style="width:145px;">Qiagen</td>
			<td style="width:119px;">28704&#160;</td>
			<td>for sequencing induced mutations</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phenol:chloroform:Isoamyl alcohol</td>
			<td style="width:145px;">Sigma</td>
			<td>P2069-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium acetate</td>
			<td style="width:145px;">Sigma</td>
			<td>S2889-250G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol (molecular biology grade)</td>
			<td style="width:145px;">Sigma</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">A9539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50X Tris-acetate-EDTA buffer</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:119px;">B49</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5-10KbRNA ladder</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:119px;">15623-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nanodrop&#160; Spectrophotometer</td>
			<td style="width:145px;">Thermo Scientific</td>
			<td style="width:119px;">Nanodrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:145px;">Sigma</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10X PBS</td>
			<td style="width:145px;">ThermoFisher</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1kb Plus DNA Ladder</td>
			<td style="width:145px;">ThermoFisher</td>
			<td>10787-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium Chloride</td>
			<td style="width:145px;">Sigma</td>
			<td>P9541-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:145px;">Sigma</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NP-40</td>
			<td style="width:145px;">ThermoFisher</td>
			<td>28324</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween 20</td>
			<td style="width:145px;">Sigma</td>
			<td>P1379-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris pH 8.3</td>
			<td style="width:145px;">Teknova</td>
			<td>T1083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">12-strip PCR tube</td>
			<td style="width:145px;">Thermo Scientific</td>
			<td style="width:119px;">AB-1113</td>
			<td></td>
		</tr>
	</tbody>
</table>

microinjection for transgenesis and genome editing in threespine sticklebacks

El pez espinoso de tres espinas se ha convertido en un potente sistema para estudiar la base genética de una amplia variedad de características morfológicas, fisiológicas y fenotipos de comportamiento. Las notablemente diversos fenotipos que han evolucionado a medida que las poblaciones marinas se adaptan a un sinnúmero de ambientes de agua dulce, junto con la capacidad de cruzar formas marinas y de agua dulce, proporcionan un sistema de vertebrados rara en la que la genética puede ser utilizado para mapear las regiones genómicas que controlan rasgos evolucionado. Excelentes recursos genómicos ya están disponibles, lo que facilita la disección genética molecular de los cambios evolucionados. Mientras que los experimentos de mapeo generar listas de genes candidatos interesantes, se requieren manipulaciones genéticas funcionales para poner a prueba las funciones de estos genes. La regulación génica puede ser estudiado con plásmidos informadores transgénicos y BACs integrados en el genoma utilizando el sistema de transposasa Tol2. Funciones de los genes candidatos específicos y cis-elementos reguladores pueden evaluarse mediante la inducción específicamutaciones con TALEN y CRISPR / Cas9 reactivos edición genoma. Todos los métodos requieren la introducción de ácidos nucleicos en fertilizados embriones stickleback una sola célula, una tarea que se hizo difícil por el espesor corion de embriones de espinosos y la relativamente pequeña y delgada de blastómeros. Aquí, un protocolo detallado para la microinyección de los ácidos nucleicos en embriones de espinosos se describe para aplicaciones transgénicas y edición de genoma para estudiar la expresión génica y la función, así como técnicas para evaluar el éxito de la transgénesis y recuperar líneas estables.

Para transgenes de genes informadores que tienen potenciador de la actividad, la inyección con éxito dará lugar a la expresión específica, celular del transgén (Figura 4A, 4C). Peces inyectados pueden ser outcrossed para producir líneas estables (ejemplo de una línea estable de BAC se muestra en la Figura 4B). La inyección de ADN en embriones de espinosos típicamente resulta en la letalidad mucho mayor que el ARN solo. Es típi...

Watch this Scientific Journal Video about Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks at JoVE.com

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

La microinyección de transgénesis y Genoma edición en tres espinas espinosos

Manipulaciones transgénicas y la edición del genoma son críticos para probar funcionalmente las funciones de los genes y elementos cis-regulador. Aquí se presenta un protocolo de microinyección detallado para la generación de modificaciones genómicas (incluyendo construcciones mediadas por Tol2 fluorescentes transgén reportero, Talens, y CRISPRs) para el modelo emergente de pescado, el espinoso de tres espinas.

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

microinjection-for-transgenesis-genome-editing-threespine

Research

JoVE Journal

Developmental Biology

13.8K vistas.  University of California, Berkeley.  Manipulaciones transgénicas y la edición del genoma son críticos para probar funcionalmente las funciones de los genes y elementos cis-regulador. Aquí se presenta un protocolo de microinyección detallado para la generación de modificaciones genómicas (incluyendo construcciones mediadas por Tol2 fluorescentes transgén reportero, Talens, y CRISPRs) para el modelo emergente de pescado, el espinoso de tres espinas. 

Video: Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

Instantánea de todo el genoma de los Reguladores de la cromatina y los Estados en<em&gt; Xenopus</em&gt; Los embriones por chip-Sec

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

Investigación

Biología del Desarrollo

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic &#8220;drivers&#8221; among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

Mosaico de pez cebra transgénesis de Análisis Genómica Funcional de candidatos genes cooperativos en la patogénesis del tumor

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Análisis de pez cebra larvas esquelético Integridad del músculo con el colorante azul de Evans

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Técnica de microinyección a Target a los países en desarrollo<em&gt; Xenopus</em&gt; riñón

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

Identificación de las condiciones críticas para inmunotinción en el guisante áfido embriones: El aumento del tejido Permeabilidad y disminución de la tinción de fondo

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

Dispositivos microestructurados para microinyección optimizado y la proyección de imagen de larvas de pez cebra

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a &#34;Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated&#34; (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

Una estrategia eficiente para la generación de sistemas de transcripción binario tejidos específicos en Drosophila genoma editando

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Sistema basado en la microinyección en Vivo implantación de cardiomiocitos embrionarios en el embrión aviar

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to their successful evolutionary radiation on the earth. The application of genetic tools such as genome editing is the key to understanding genetic changes that are responsible for evolutionary transitions in an Evo-Devo approach. In this article, we describe our current protocol for generating and maintaining mutant strains of T. domestica. We report a dry injection method, as an alternative to the reported wet injection method, that allows us to obtain stably high survival rates in injected embryos. We also report an optimized environment setting to mate adults and obtain subsequent generations with high efficiency. Our method underlines the importance of taking each species&#8217; unique biology into account for the successful application of genome editing methods to non-traditional model organisms. We predict that these genome editing protocols will help in implementing T. domestica as a laboratory model and to further accelerate the development and application of useful genetic tools in this species.

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

We provide a detailed protocol for rearing, microinjection of eggs and for efficient mating of the firebrat Thermobia domestica to generate and maintain mutant strains after genome editing.

Microinyección de óvulos y apareamiento eficiente para la edición del genoma en la thermobia doméstica de firebrat

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

Generación de organoides cerebrales a partir de células madre pluripotentes inducidas en mini biorreactores caseros

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

La microinyección de transgénesis y Genoma edición en tres espinas espinosos

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