Agradecemos al Dr. Doris K. Wu de plásmidos de expresión y sondas in situ, el Centro de la Universidad Johns Hopkins para la instalación de imágenes Biología Sensorial y el Centro para la audición y el equilibrio. Este trabajo fue apoyado por NIDCD subvención T32 DC000023 de LE

Los huevos no eclosionados y embriones son atendidos y tratados con ética y humanamente. Todos los protocolos para el uso de embriones sin eclosionar fueron aprobados por el Cuidado de Animales y el empleo en la Escuela de Medicina Johns Hopkins, Baltimore, Maryland. 1. Los huevos y la preparación de construcciones de expresión <ol><li> Huevos: <ol><li> Incubar 3 - 4 docenas de huevos de gallina fertilizados (Gallus gallus) acostado sobre sus lados a 37 ° C. <ol><li> Después de 24 horas de incubación, tirar de 3 cc de albúmina con una jeringa desde el extremo posterior redonda del huevo y sellar el agujero con cinta adhesiva transparente. Esto crea una cámara de aire entre el embrión y la cáscara del huevo, lo que permite la facilidad de acceso al embrión sin el embrión se adhiera a la shell cuando el corte de la ventana para el acceso en la parte superior del huevo 19. </li><li> En 117 horas de tiempo de incubación, los embriones etapa de acuerdo con 20 etapas de desarrollo Hamburger y Hamiltonen E4 (HH24-25) (Ver Figura 1D). </li></ol></li></ol></li><li> Construcciones de expresión: <ol><li> Preparar el DNA plásmido usando reactivos y el protocolo proporcionado por el fabricante de un kit de preparación disponible comercialmente. En la etapa final de preparación, eluir el ADN en 500 l de TE (Tris-EDTA) tampón proporcionado en el kit en un tubo de 1,5 ml. </li><li> El etanol precipitar el ADN para concentrarla mediante la adición de 50 l de 3 M de acetato de sodio y 1 ml de etanol 100% al tubo. Se coloca el tubo a -20 ° CO / N. </li><li> Centrifugar el tubo durante 30 min a 14.000 rpm a 4 ° C. Eliminar el sobrenadante y aire secar el sedimento durante 10 min a RT. Disolver el precipitado en 15 l de tampón TE, lo que debería dar una concentración de ~ 4 mg / l. NOTA: El control de construcciones de expresión utilizados aquí son PME-IRES-GFP 10, que es impulsado por un promotor de pollo β-actina, o PCI-H2B-IRES-RFP <shasta&gt; 10, que es impulsado por el promotor CMV. </li></ol></li></ol> 2. pollo in ovo Micro-electroporación in ovo y ensayo de proliferación celular <ol><li> En Ovo Microinyección de construcción de expresión y Micro-electroporación: <ol><li> Tire de los tubos capilares de vidrio en agujas finas usando un extractor de micropipeta con los siguientes parámetros optimizados para una 2,5 x 2,5 mm Box Platinum Calefacción Filament: Presión = 500, Heat = 600 ° C, Pull = 64, velocidad = 105, y la hora = 150 ms (ver Tabla 1). </li><li> Establecieron la izquierda y etapas micromanipulador diestros que flanquean el microscopio (véase la Figura 1A). El micromanipulador Dretà sostiene la aguja capilar de vidrio y el micro-manipulador zurdos sostiene los electrodos (Figura 1A). </li><li> Preparar la aguja capilar de vidrio finamente tirado por microinyección cortandola punta de la aguja con un fórceps durante el trabajo bajo el microscopio. </li><li> Llene la aguja de forma manual a mano utilizando el micromanipulador con 2,5 l de ADN plásmido teñidas con 0,1% Fast Green mientras se trabaja bajo el microscopio. </li><li> Colocar el huevo acostado de lado dentro de un dispositivo de retención de moldes / huevo (ver Figura 1A). Cortar una ventana redonda en la parte superior del huevo con unas tijeras 19 (Figura 1A). </li><li> Mientras que trabajar bajo el microscopio, abra con cuidado las dos membranas superpuestas el embrión utilizando pinzas. </li><li> Mientras que trabajar bajo el microscopio, entregar aproximadamente ~ 0,5 l de ADN plásmido a la derecha del lumen de la vesícula ótica por microinyección de forma manual utilizando la mano derecha utilizando el micro-manipulador y por micro-electroporación. <ol><li> Para ello, micro-inyectar el derecho lumen vesícula ótica con el ADN con la mano derecha micromanipulador (Figura 1B-D), mientras que al mismo tiempo usando la mano izquierda aguantandoga pinza de sostener la cabeza del embrión, porque la cabeza del embrión se sumerge en la etapa E4. No te micro-inyectar más allá del lumen de la vesícula ótica, ya que esto dañará la vesícula ótica. </li><li> Inmediatamente después de la microinyección, mientras se trabaja bajo el microscopio, usar de la izquierda micro-manipulador para colocar el positivo (ánodo) 2 mm electrodo de platino anterior-ventral a la vesícula ótica derecho y el electrodo 2 mm negativo (cátodo) en paralelo 1 mm entre sí (véase la Figura 1 C). Entregar 4 pulsos a 12 V con 100 ms de duración y 200 mseg separación. La vesícula ótica izquierda sirve como un control no tratado interna. NOTA: Limpie los electrodos en el medio electroporaciones con un bastoncillo de algodón empapado en 1x PBS. </li></ol></li></ol></li><li> En Ovo ensayo de proliferación celular: <ol><li> Inmediatamente después de la electroporación se añaden 50 l de 0,25 mg / ml en 1 x PBS EdU dejando caer manualmente la solución EdU 50 l en el conjuntoembrión in ovo utilizando una pipeta. Sellar los huevos con la cinta y volver a la incubadora durante 18 a 96 hr. </li><li> Retirar con cuidado la cinta y comprobar los embriones para GFP señal fluorescente o RFP dentro de la vesícula ótica después de 18 - 24 horas usando un microscopio fluorescente estándar (ver Figura 1 EE &#39;). Si la señal fluorescente está presente, en este punto cosechar los embriones de los análisis o vuelva a sellar con cinta y devolverlo a la incubadora para la recolección y análisis en etapas posteriores (ver Figura 1 FP &#39;&#39;). NOTA: La expresión de GFP señal con la construcción PME-IRES-GFP es evidente 6 - 7 horas después de la electroporación 10. </li></ol></li></ol> 3. La recolección de embriones y Procesamiento de tejidos para la ARN hibridación in situ e inmunohistoquímica <ol><li> Recoger los embriones utilizando fórceps. Enjuague el embrión en 1x PBS frío. Retire las cabezas de los embriones, cortando la cabeza por el cuelloel uso de fórceps, y colocar las cabezas en paraformaldehído al 4% O / N a 4 ° C. Perforar el cerebro utilizando una pinza para permitir la penetración de la paraformaldehído 4% dentro de la cabeza. </li><li> Deshidratar las cabezas en sacarosa al 30% (en PBS 1x) O / N a 4 ° C. </li><li> Montar las cabezas en medio crioprotector mediante congelación rápida en una suspensión de hielo seco y metilbutano (2-metilbutano). Por otra parte, la rápida congelación de las cabezas en medio crioprotector con nitrógeno líquido. Almacenar las cabezas montadas a -80 ° C. </li><li> Cryosection las cabezas en 12 micras de espesor secciones de tejidos y recoger todas las secciones de tejido que contiene el oído interno-en portaobjetos de vidrio de microscopio superfrosted. </li><li> Realizar RNA estándar de hibridación in situ e inmunohistoquímica, como se describe previamente 4,21, y analizar para la proliferación celular EdU siguiendo el protocolo proporcionado por el fabricante del kit. NOTA: Las células ciliadas se pueden identificar usando policlonales de conejo α-myosinVIIA (MYO7A) unatibody (1: 1000, Proteus Biosciences) y anticuerpo secundario marcado con fluorescencia (1: 250, de cabra anti-conejo AlexaFluor 488; Invitrogen). </li></ol> 4. Captura y Procesamiento <ol><li> Tomar imágenes con equipos de imagen digital. Resultados de inmunohistoquímica imagen con un microscopio de fluorescencia estándar y los resultados de la in situ usando un microscopio de campo amplio estándar (que van de 5x a 40x en aumento). </li><li> Procesar las imágenes utilizando el software de procesamiento de imágenes. </li></ol>

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The authors have nothing to disclose.

El método aquí descrito de in ovo micro-electroporación se ha optimizado para la transferencia de genes en el órgano auditivo en desarrollo. Es compatible con vectores de expresión basados ​​en el ADN de plásmido normalmente se utilizan para manipular la función del gen / expresión. El momento de la electroporación a E4 es óptimo para las investigaciones que se centran en el desarrollo de HC en el órgano auditivo. Los pasos más críticos son micro-inyección de la DNA en el lumen vesícula ótic...

A pesar de las diferencias en la morfología celular y patrones entre los mamíferos y órgano sensorial auditivo aviar, se cree que están conservadas evolutivamente 1-3 los factores moleculares y las vías responsables del desarrollo de HC sensorial. La papila basilar, que aloja la HC auditivas y sus alrededores SC, se desarrolla como la protrusión de la otocyst oído interno. Al principio de un grupo de progenitores de HC y SC se especifica dentro de la placoda ótica / Dominio competente taza neuronal sensorial-ótica (NSD). Los datos de mapeado de destino proporcionan evidencia de que los linajes neuronales y sensoriales están vinculados y se derivan de la NSD situado en la región antero-ventral de la copa ótica o otocyst en ratones y pollos 4,5. En primer lugar, los neuroblastos delaminate de la NSD para dar lugar a las neuronas del ganglio auditivo-vestibular, que finalmente dividido en ganglios auditivo y vestibular. Estas neuronas inervan los HC sensoriales del oído interno y núcleos en el tronco cerebral. Las células Tha permanecer en el NSD se cree que dar lugar a varios parches sensoriales, incluido el órgano sensorial auditivo, que consta de los HC sensoriales mecano-transducción y sus asociados SCs. En tanto el pollo y murino, órgano de la audición sensorial progenitora salida del ciclo celular y la diferenciación HC se producen en gradientes opuestos. En polluelo, la salida del ciclo celular progenitora auditiva comienza alrededor de ~ E5 y progresa desde la base hasta el vértice, y desde el centro hacia la periferia 6. Un día después, HC diferenciación comienza en el vértice y que avanza a la base 7. Estudiar el desarrollo del oído interno en el pollo proporciona muchas ventajas técnicas como mecanismos funcionales pueden ser investigados con relativa facilidad in ovo en comparación con la manipulación de los embriones en el útero en otros sistemas modelo, que requiere cirugías complejas. El método descrito aquí utiliza in ovo micro-electroporación para apuntar genes de interés en el Basila presuntivar área de la papila antero-ventral en la vesícula ótica específicamente a E4. La electroporación in ovo es una técnica que está bien establecido y comúnmente utilizado 8-14. El principio de la técnica de electroporación se basa en el hecho de que los nucleótidos (por ejemplo, ADN plásmido, ADN sintético, o ARN oligonucleótidos) están cargadas negativamente. El ADN se inyecta en el tejido de interés. Cuando una corriente eléctrica se coloca a través del tejido, la corriente abre los poros transitorios en las paredes celulares y permite la captación del ADN, como fluye el ADN cargado negativamente hacia el electrodo positivo (ánodo). Para los experimentos de ganancia de función, los genes de interés se subclonan comúnmente en plásmidos de expresión que contienen casetes de expresión adecuados para la proteína verde fluorescente (GFP) o la proteína fluorescente roja (RFP). Para los experimentos de construcciones de pérdida de función basados ​​en plásmidos dominantes negativos represores, o RNAi, o construcciones de morfolino son commonly utiliza 15,16. Para inhibir microARN (miARN) de función miRNAs construcciones de esponja, que son típicamente plásmido basado, se puede utilizar 17. El método aquí descrito permite la investigación de los mecanismos moleculares que controlan la proliferación y diferenciación en el órgano auditivo, como el método en micro-electroporación in ovo se puede combinar fácilmente con en ensayo de proliferación celular in ovo mediante la adición de EdU a todo el embrión. La electroporación y la adición de EdU se realiza a E4 en la vesícula ótica, que es un día antes del inicio de la salida del ciclo celular en el órgano auditivo. Análisis del órgano de la audición se realiza típicamente 18 de - 96 horas después de la electroporación en E5 (inicio de la salida del ciclo celular progenitora sensorial), E6 (inicio de la diferenciación HC), y E7 (durante la diferenciación HC); y más tarde hasta ~ E9 (final de la diferenciación HC). Este método de electroporación de la transferencia genética es transitoria que dura aproximadamente ~ 4 días, because los genes de interés no se integran en el genoma, pero el método es aplicable para su uso con vectores de expresión basados ​​en el ADN de plásmido apropiado, que tiene la capacidad de integrarse en el genoma, tales como la transferencia de genes mediada por Tol2 11. Con este método robusto GFP o RFP expresión dura ~ 4 días en la cóclea, después de lo cual las señales se desvanecen, sin embargo, proporcionan una ventana de tiempo suficiente para estudiar la intrincada desarrollo del órgano auditivo. Esto, a su método de transferencia génica in ovo es novedosa y permite apuntar específicamente el órgano auditivo presuntiva en E4, que es óptima para las investigaciones que se centran en el desarrollo de HC en el órgano auditivo. Es una buena adición a procedimientos alternativos, que electroporate en etapas de desarrollo mucho más jóvenes o 11,12 en comparación con el uso de cultivos de explantes in vitro papilas basilar 18.

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gene transfer into the chicken auditory organ by in ovo micro-electroporation

Embriones de pollo son sistemas modelo ideal para estudiar el desarrollo embrionario como manipulaciones de la función de genes pueden llevarse a cabo con relativa facilidad in ovo. El órgano sensorial auditivo del oído interno es fundamental para nuestra capacidad de oír. Alberga un epitelio sensorial altamente especializado que consiste en células ciliadas mecano-transducción (HC) y que rodea la glía como células de soporte (SCS). A pesar de las diferencias estructurales en los órganos auditivos, los mecanismos moleculares que regulan el desarrollo del órgano auditivo se conservan evolutivamente entre los mamíferos y aves En electroporación in ovo está muy limitado a las primeras etapas de E1 -. E3. Debido al desarrollo relativamente tarde del órgano auditivo en E5, manipulaciones del órgano de la audición por electroporación in ovo en el pasado E3 son difíciles debido al avanzado desarrollo del embrión de pollo en etapas posteriores. El método que aquí se presenta es un método de transferencia génica transitoria para dirigir genes de interés enetapa E4 - E4.5 en el auditorio de pollo en desarrollo a través de los órganos sensoriales in ovo micro-electroporación. Este método es aplicable para ganancia y la pérdida de funciones con vectores de expresión basados ​​en el ADN de plásmido convencional y se puede combinar con en ensayo de proliferación celular in ovo mediante la adición de EdU (5-etinil-2 &#39;desoxiuridina) a todo el embrión en el tiempo de electroporación. El uso de la proteína fluorescente verde o roja (GFP o RFP) plásmidos de expresión permite al experimentador para determinar rápidamente si la electroporación dirigido con éxito la porción auditiva del oído interno en desarrollo. En este trabajo el método, se ilustran ejemplos representativos de las muestras GFP a electroporación; embriones se recogieron 18 a 96 h después de la electroporación y la selección de GFP a la zona de pro-sensorial del órgano auditivo se confirmó mediante hibridación in situ de ARN. El papel método también proporciona un protocolo optimizado para el uso de la timidina analógico EdU para analizar prolife célularación; Se proporciona un ejemplo de un ensayo de proliferación celular basado EdU que combina inmuno-etiquetado y haga clic en la química edu.

En este ADN método de papel plásmido que consiste en proteína fluorescente verde (GFP) casetes de expresión fue el blanco en el pollo en desarrollo papila basilar (BP) con parámetros optimizados de 12 V y 4 pulsos con 100 duración de pulso mseg y los intervalos de 200 mseg, produciendo un ~ 50% de tasa de supervivencia de los embriones y la eficiencia de plásmidos de ADN de direccionamiento en el BP. De imagen fluorescente de la expresión de GFP nativa en el desarrollo de embrion...

Watch this Scientific Journal Video about Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation at JoVE.com

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

La transferencia de genes en el pollo auditiva de órgano de<em&gt; En Ovo</em&gt; Micro-electroporación

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

gene-transfer-into-chicken-auditory-organ-ovo-micro

Research

JoVE Journal

Developmental Biology

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

9.4K vistas.  The Johns Hopkins University, School of Medicine. The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ. 

Video: Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Cultura de embriones de ratón explantes cocleares y Gene Transfer por electroporación

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Investigación

Biología del Desarrollo

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

En Electroporations Ovo de embriones de pollo HH Etapa 10

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Biología

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

En Ovo electroporación en el tronco encefálico auditivo del pollo

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Transferencia de genes a la oreja del ratón en desarrollo por el interior<em&gt; En Vivo</em&gt; La electroporación

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Neurociencias

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

En electroporación in ovo en el mesencéfalo polluelo para estudiar la función génica en el desarrollo neuronal dopaminérgica

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

En Expression in ovo de microARN en ventral del polluelo Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

Métodos In Ovo y Ex Ovo para estudiar el desarrollo del oído interno aviar

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

En electroporación in ovo en la retina de embriones de pollo

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Transferencia de genes en embriones de pollo por los mayores<em&gt; Ex ovo</em&gt; La electroporación

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

La transferencia de genes en el pollo auditiva de órgano de

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