Agradecemos ao Dr. Doris K. Wu por plasmídeos de expressão e sondas in situ, o Centro Universitário Johns Hopkins para a instalação de imagem Biologia Sensorial e do Centro de Audição e Equilíbrio. Este trabalho foi apoiado por NIDCD Grant T32 DC000023 a LE

Os ovos e embriões não eclodidos são cuidadas e tratadas de forma ética e humana. Todos os protocolos de utilização de embriões unhatched foram aprovados pelo Animal Care e do Comitê Use na Johns Hopkins School of Medicine, em Baltimore, Maryland. 1. Os ovos e Preparação de construções de expressão <ol><li> Ovos: <ol><li> Incubar 3-4 dúzia de ovos de galinha fertilizados (Gallus gallus) deitado em seus lados a 37 ° C. <ol><li> Após 24 horas de incubação, puxe 3 cc de albumina com uma seringa a partir da extremidade traseira rodada do ovo e selar o buraco com fita adesiva transparente. Isto cria uma câmara de ar entre o embrião e a casca do ovo, permitindo a facilidade de acesso para o embrião sem o embrião que adere ao invólucro de corte quando a janela de acesso no topo do ovo 19. </li><li> Em 117 horas de tempo de incubação, encenar os embriões de acordo com Hamburger e Hamilton 20 estágios de desenvolvimentoa E4 (HH24-25) (ver figura 1D). </li></ol></li></ol></li><li> Expressão Constrói: <ol><li> Prepara-se o ADN do plasmídeo usando reagentes e o protocolo fornecido pelo fabricante do kit de uma preparação disponível comercialmente. No passo final da preparação, eluir o ADN em 500 ul de TE (Tris-EDTA) tampão fornecido no kit para um tubo de 1,5 ml. </li><li> Etanol precipitar o ADN para concentrá-la por adição de 50 ul de acetato de sódio 3M e 1 ml de etanol a 100% ao tubo. Colocar o tubo à temperatura de -20 ° CO / N. </li><li> Centrifuga-se o tubo durante 30 minutos a 14000 rpm a 4 ° C. Remover o sobrenadante e ar-secar o sedimento durante 10 minutos à TA. Dissolve-se o sedimento em 15 ul de tampão TE, os quais devem produzir uma concentração de ~ 4 mg / mL. NOTA: O construções de expressão de controlo utilizados aqui são PMES-IRES-GFP, 10, que é conduzido por um promotor de frango β-actina, ou pCI-H2B-IRES-RFP <saté&gt; 10, que é conduzida pelo promotor de CMV. </li></ol></li></ol> 2. galinha In Ovo Micro-electroporação e In Ovo celular Ensaio de Proliferação <ol><li> Em Ovo Micro-injecção de construção de expressão e Micro-eletroporação: <ol><li> Retire os tubos capilares de vidro em agulhas finas utilizando um extractor de micropipeta com os seguintes parâmetros optimizados para um 2,5 x 2,5 milímetros Caixa de platina de aquecimento de filamento: Pressão = 500, de calor = 600 ° C, Pull = 64, velocidade = 105, e Tempo = 150 mseg (ver Tabela 1). </li><li> Configure a esquerda e para a fase micro-manipulador destros flanqueando o microscópio (ver Figura 1A). O micro-manipulador Destro segura a agulha capilar de vidro e micro-manipulador canhota detém os eletrodos (Figura 1A). </li><li> Prepare a agulha finamente puxado capilar de vidro para micro-injecção, cortandoa ponta da agulha com uma pinça, enquanto trabalha com o microscópio. </li><li> Encher a agulha manualmente à mão usando o micro-manipulador com 2,5 ul de ADN de plasmídeo coloridas com 0,1% de Fast Green, enquanto que trabalham sob o microscópio. </li><li> Coloque o ovo deitado de lado dentro de um dispositivo de suporte para moldes / ovo (ver Figura 1A). Cortar uma janela redonda na parte superior do ovo usando uma tesoura 19 (Figura 1A). </li><li> Enquanto trabalhava sob o microscópio, abra com cuidado as duas membranas que recobrem o embrião usando uma pinça. </li><li> Ao trabalhar sob o microscópio, entregar aproximadamente ~ 0,5 ul de ADN de plasmídeo para o lúmen vesícula ótica direita por micro-injecção manualmente utilizando a mão direita, utilizando o micro-manipulador e por micro-electroporação. <ol><li> Para tal, a micro-injectar o lúmen vesícula ótica direita com o ADN com o lado direito do micro-manipulador (Figura 1B-D), ao mesmo tempo usando a mão esquerda Holdinga pinça para segurar a cabeça do embrião, porque a cabeça do embrião mergulha na fase E4. Não micro-injetar passado o lúmen vesícula ótica, porque isso irá prejudicar a vesícula ótica. </li><li> Imediatamente após a micro-injecção, enquanto trabalhava sob o microscópio, use a Esquerda mão micro-manipulador para colocar o positivo (ânodo) 2 milímetros eletrodo de platina ântero-ventral à vesícula ótica direita e o eletrodo de 2 milímetros negativo (cátodo) em paralelo 1 mm entre si (ver Figura 1 C). Deliver 4 impulsos a 12 V com 100 mseg de duração e espaçamento 200 mseg. A vesícula ótica esquerda serve como um controlo não tratado interno. NOTA: Limpe os eléctrodos entre electroporations com um chumaço de algodão embebido em 1x PBS. </li></ol></li></ol></li><li> In Ovo celular Ensaio de Proliferação: <ol><li> Imediatamente após a electroporação adicionar 50 uL de 0,25 mg / mL em 1X PBS EdU soltando manualmente a solução EdU 50 ul para o conjuntoembrião no ovo usando uma pipeta. Selar os ovos com fita e retornar para o incubador durante 18-96 horas. </li><li> Remova cuidadosamente a fita e verifique os embriões para GFP fluorescente ou sinal de RFP dentro da vesícula ótica após 18-24 horas usando um microscópio de fluorescência padrão (veja a Figura 1 EE &#39;). Se o sinal fluorescente está presente, neste ponto colher os embriões para análise ou feche com fita adesiva e devolvê-lo para a incubadora para a colheita e analisa em fases posteriores (ver Figura 1 FP &#39;&#39;). NOTA: Expressão de sinal de GFP com o constructo PMES-IRES-GFP é evidente 6-7 horas após a electroporação 10. </li></ol></li></ol> Colheita 3. Embryo e processamento de tecidos para o RNA hibridização in situ e imuno-histoquímica <ol><li> Colher os embriões usando uma pinça. Lavar o embrião em 1x PBS frio. Retirar as cabeças dos embriões, cortando a cabeça por seu pescoçousando uma pinça e coloque as cabeças em paraformaldeído a 4% O / N a 4 ° C. Perfurar o cérebro utilizando um fórceps para permitir a penetração do paraformaldeído 4% dentro da cabeça. </li><li> Desidratar as cabeças em 30% de sacarose (em 1x PBS) O / N a 4 ° C. </li><li> Montar as cabeças em forma crioprotector por congelação rápida em uma suspensão de gelo seco e metilbutano (2-metilbutano). Alternativamente, rápida congelar as cabeças em meio crioprotector com nitrogênio líquido. Armazenar as cabeças montadas à temperatura de -80 ° C. </li><li> Criocorte as cabeças em 12 mm cortes de tecidos grossos e recolher todas as secções de tecido contendo ouvido interno em lâminas de microscópio de vidro superfrosted. </li><li> Realizar ARN padrão de hibridação in situ e imuno-histoquímica como previamente descrito 4,21, analisar e para a proliferação celular EdU seguindo o protocolo fornecido pelo fabricante do kit. NOTA: As células ciliadas podem ser identificadas usando policlonais de coelho α-MyosinVIIa (MYO7A) umtibody (1: 1000, Proteus Biosciences) e o anticorpo secundário marcado fluorescentemente (1: 250, de cabra anti-coelho AlexaFluor 488; Invitrogen). </li></ol> 4. Captura e Processamento de Imagem <ol><li> Tire fotos com equipamentos de imagem digital. Resultados de imunohistoquímica imagem com um microscópio de fluorescência padrão e os resultados dos controlos no local utilizando um microscópio de campo amplo padrão (que varia de 5x a 40x em ampliação). </li><li> Processar as imagens usando o software de processamento de imagem. </li></ol>

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The authors have nothing to disclose.

O método aqui descrito de in ovo micro-electroporação é optimizado para a transferência de genes para o órgão auditivo em desenvolvimento. É compatível com vectores de expressão baseados em DNA de plasmídeo tipicamente usadas para manipular a função do gene / expressão. O momento da eletroporação na E4 é o ideal para as investigações que incidem sobre o desenvolvimento HC no órgão auditivo. Os passos mais críticos são micro-injecção do ADN para o lúmen vesícula ótica sem ir muito pro...

Apesar das diferenças na morfologia e padronização celular entre os mamíferos e órgão sensorial auditiva aviária, os fatores moleculares e vias responsáveis ​​pelo desenvolvimento HC sensorial são pensados ​​para ser evolutivamente conservada 1-3. A papila basilar, que abriga os HCs auditivas e suas SCs circundantes, se desenvolve como um outpocketing do otocyst ouvido interno. Logo no início de uma piscina de progenitores HC e SC é especificado dentro do placï¿½io ótica ótica domínio / copo neural-sensorial competente (NSD). Dados destino de mapeamento de fornecer evidências de que linhagens neuronais e sensoriais são ligados e surgem da NSD localizada na região antero-ventral do copo ótica ou otocyst em ratos e galinhas 4,5. Em primeiro lugar, neuroblasts delaminate da NSD para dar origem aos neurônios do gânglio auditivo-vestibular, o que acabou dividida em gânglios auditivo e vestibular. Esses neurônios inervam os HCs sensoriais do ouvido interno e núcleos no tronco cerebral. As células Them permanecer no NSD são pensados ​​para dar origem a várias manchas sensoriais, incluindo o órgão auditivo sensorial, que consiste em os HCs sensoriais de transdução mecano e seus SCs associados. Em ambos os pintos e murino, órgão auditivo progenitor sensorial saída do ciclo celular e diferenciação HC ocorrer em gradientes opostos. Em pintinho, progenitor auditiva saída do ciclo celular começa em torno de ~ E5 e progride a partir da base para o ápice, e do centro para a periferia 6. Um dia mais tarde, começa HC diferenciação no vértice e progride para a base 7. Estudando o desenvolvimento do ouvido interno em frango proporciona muitas vantagens técnicas como mecanismos funcionais podem ser investigadas com relativa facilidade in ovo em oposição a manipulação de embriões no útero, em outros sistemas de modelos, que requer cirurgias complexas. O método descrito aqui utiliza in ovo micro-electroporação de genes alvo de interesse no Basila presuntivoárea de papila r ântero-ventral na vesícula ótica especificamente a E4. A electroporação in ovo é uma técnica que está bem estabelecida e utilizada 8-14. O princípio da técnica de electroporação, é baseado no facto de nucleótidos (por exemplo, ADN de plasmídeo, DNA sintético, RNA ou oligonucleótidos) são carregadas negativamente. O ADN é injectada no tecido de interesse. Quando uma corrente eléctrica é colocada ao longo do tecido, a actual abre poros transientes nas paredes das células e permite a captação do ADN, como flui do ADN carregado negativamente em direcção ao eléctrodo positivo (ânodo). Para as experiências de ganho-de-função, os genes de interesse são subclonados em vulgarmente plasmídeos de expressão que contêm cassetes de expressão adequados para a proteína verde fluorescente (GFP) ou a proteína fluorescente vermelha (RFP). Para as experiências de construções de perda de função com base em plasmídeos dominantes negativos repressores, ou RNAi, ou construções morfolino são commonly usado 15,16. Para inibir microARN (miARN) função miARNs construções de esponja, que são tipicamente plasmídeo com base, pode ser usado 17. O método aqui descrito permite investigar os mecanismos moleculares que controlam a proliferação e diferenciação no órgão auditivo, em que o método de micro-electroporação in ovo pode ser facilmente combinado com no ensaio de proliferação celular in ovo adicionando EdU a todo o embrião. A electroporação e a adição de EdU é realizada em E4 na vesícula ótica, que é um dia antes do início da saída do ciclo celular no órgão auditivo. Análise do órgão auditivo é tipicamente realizada 18-96 horas após a electroporação em E5 (aparecimento de células progenitoras saída sensorial do ciclo celular), E6 (início da diferenciação HC), e E7 (durante a diferenciação HC); e mais tarde até ~ E9 (fim da diferenciação HC). Este método de electroporação de transferência de genes é transitória que dura aproximadamente 4 dias ~, because os genes de interesse não se integram no genoma, mas o método é aplicável para utilização com vectores de expressão à base de ADN de plasmídeo apropriado, que têm a capacidade de integrar no genoma, tal como a transferência de genes mediada Tol2-11. Com este método GFP robustos ou expressão RFP dura ~ 4 dias na cóclea, após o que apontam os sinais desaparecem, mas fornecem uma janela de tempo suficiente para estudar o desenvolvimento intrincado do órgão auditivo. Este, por método de transferência de genes in ovo é novo e permite que visar especificamente o órgão auditivo presuntivo em E4, que é ideal para as investigações que incidem sobre o desenvolvimento HC no órgão auditivo. É um bom complemento para métodos alternativos, que electroporate em muito mais jovens estágios de desenvolvimento 11,12 ou em comparação com o uso de in vitro papilas basilar de explantes culturas 18.

<table><tbody><tr><td>Sterile 1x PBS pH 7.4</td><td>gibco</td><td>10010-023</td><td></td></tr><tr><td>Fast Green FCF Powder</td><td>Sigma</td><td>F7252-5G</td><td></td></tr><tr><td>EdU Powder</td><td>Invitrogen</td><td>E10187</td><td></td></tr><tr><td>Click-IT EdU Alexa Fluor 555 Imaging Kit</td><td>Invitrogen</td><td>C10338</td><td></td></tr><tr><td>HiSpeed Plasmid Midi Kit (25)</td><td>Qiagen</td><td>12643</td><td></td></tr><tr><td>ECM 830 ElectroSquarePorator&amp;nbsp;</td><td>BTX Harvard Apparatus</td><td></td><td></td></tr><tr><td>Banana to Micrograbber Cable Kit</td><td>BTX Harvard Apparatus</td><td>45-0216</td><td></td></tr><tr><td>Right Handed &amp;amp; Left Handed Micromanipulators</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>M3301R &amp;amp; M3301L</td><td></td></tr><tr><td>Two 12 mm Magnetic Holding Device Stages with 7 inch vertical posts</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>M10</td><td></td></tr><tr><td>Metal Steel Base Plate 12x24 inch</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>5479</td><td></td></tr><tr><td>Scissors for Eggs 12 cm long curved, 12 mm extrafine blades; Spring Scissors</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>14120</td><td></td></tr><tr><td>Micropippette Puller for pulling needles</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>2.5x2.5 mm Box Platinum Heating Filament</td><td>Sutter Instrument Co.</td><td></td><td></td></tr><tr><td>Glass Capillary Tubes/Needles/No Fiber/Borosil 1 mm</td><td>FHC</td><td>27-30-0</td><td></td></tr><tr><td>Hamilton Glass Syringe 100 &amp;mu;l</td><td>Hamilton</td><td>80601 Model 710LT</td><td></td></tr><tr><td>Mineral Oil (Heavy) for Hamilton Glass Syringe</td><td>Fisher Scientific</td><td>O122-1</td><td></td></tr><tr><td>Polyethylene Tubing for connecting the Glass Syringe and Glass Cappillary Needles/ Non Toxic</td><td>Becton Dickinson and Company (BD)</td><td>427420 Intramedic Clay Adams Brand</td><td></td></tr><tr><td>3 cc Disposable Syringes</td><td>Becton Dickinson and Company (BD)</td><td>309657</td><td></td></tr><tr><td>Disposable 21 Gauge Needles</td><td>Becton Dickinson and Company (BD)</td><td>305122</td><td></td></tr><tr><td>One pair of 2 mm Platinum Electrodes</td><td>Bulldog Bio. / Nepagene</td><td>CUY611P3-2</td><td></td></tr><tr><td>Electrode Holder</td><td>Bulldog Bio. / Nepagene</td><td>CUY580</td><td></td></tr><tr><td>One pair of Dumont fine forceps number 5</td><td>Fine Science Tools (FST)</td><td></td><td></td></tr><tr><td>Matte finish invisible tape for sealing eggs</td><td>Office Depot</td><td>520-928</td><td></td></tr><tr><td>Cotton-Tipped Swabs</td><td>Fisher Scientific</td><td>23-400-101</td><td></td></tr><tr><td>Sterile filter tips</td><td></td><td></td><td></td></tr></tbody></table>

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

Embriões de galinha são sistemas modelo ideal para estudar o desenvolvimento embrionário como manipulações da função do gene pode ser realizada com relativa facilidade in ovo. O ouvido interno órgão sensorial auditivo é fundamental para a nossa capacidade de ouvir. Ele abriga um epitélio altamente especializada sensorial que consiste em células ciliadas de transdução mecano (HCs) e envolvente glial-como células de suporte (SCS). Apesar das diferenças estruturais nos órgãos auditivos, os mecanismos moleculares que regulam o desenvolvimento do órgão auditivo são evolutivamente conservada entre mamíferos e aves Em electroporação in ovo é largamente limitado a fases precoces em E1 -. E3. Devido ao desenvolvimento tardio relativa do órgão auditivo na E5, manipulações do órgão auditivo por em eletroporação in ovo passado E3 são difíceis devido ao desenvolvimento avançado do embrião de galinha em fases posteriores. O método aqui apresentado é um método de transferência de genes transitória para alvejar genes de interesse noestágio E4 - E4.5 no frango auditivo órgão sensorial desenvolvimento via in ovo micro-eletroporação. Este método é aplicável para gain- e perda de funções com vectores de expressão baseados em DNA de plasmídeo convencional e pode ser combinada com a proliferação celular num ensaio in ovo adicionando EdU (5-etinil-2&#39;-desoxiuridina) para todo o embrião no tempo de electroporação. O uso da proteína fluorescente verde ou vermelho (GFP ou RFP) plasmídeos de expressão permite que o experimentador para determinar rapidamente se a eletroporação alvo com sucesso a porção auditiva do ouvido interno em desenvolvimento. Neste trabalho método, exemplos representativos dos espécimes GFP electroporadas são ilustrados; embriões foram recolhidas 18-96 horas após a electroporação e a segmentação de GFP à região pró-sensitivo do órgão auditivo foi confirmada por RNA de hibridação in situ. O papel método também proporciona um protocolo optimizado para a utilização da timidina EdU analógico para analisar célula prolifração; um exemplo de um ensaio de proliferação celular baseado EdU que combina imuno-marcação e clique EdU química é fornecida.

Neste ADN método papel plasmídeo consistindo de proteína fluorescente verde (GFP) cassetes de expressão foi alvo para o desenvolvimento de frango papila basilar (PA), com parâmetros optimizados de 12 V e 4 impulsos com 100 duração do impulso mseg e intervalos de 200 mseg, obtendo-se uma ~ taxa de sobrevivência embrionária 50% e eficiência de plasmídeo de DNA alvo para a BP. Imagiologia de fluorescência da expressão da GFP nativa no desenvolvimento de embriões mostraram este...

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

A transferência de genes para o frango auditivo órgão por<em&gt; In Ovo</em&gt; Micro-eletroporação

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 visualizações.  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. 

Vídeo: 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 da embrionárias mouse cocleares explantes e Gene Transferência de Eletroporação

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

Biologia do Desenvolvimento

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.

Em Electroporations Ovo de Estágio HH 10 embriões de galinha

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 ...

Biologia

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.

Em Ovo Electroporation no Brainstem Auditivo da Galinha

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.

Transferência de genes para a orelha do camundongo Desenvolvimento Inner por<em&gt; In Vivo</em&gt; Eletroporação

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 ...

Neurociência

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.

Na eletroporação in ovo em Midbrain pintainho para estudar a função do gene em dopaminérgico Desenvolvimento Neuron

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.

Em Expression in ovo de microRNA em Ventral Pintinho 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.

In Ovo e Ex Ovo Métodos para estudar o desenvolvimento do ouvido interno aviário

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.

Em Eletroporação Ovo na retina de pinto embrionário

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.

Transferência de Embriões em Gene de frango por mais velhos<em&gt; Ovo ex</em&gt; Eletroporação

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.

A transferência de genes para o frango auditivo órgão por

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