This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology to M.I. (21229004 and 25221304).

O uso de ratos para a investigação estava em conformidade com as orientações estabelecidas pelo Comitê da Universidade de Tóquio Animal Welfare. 1.Preparation de DNA de plasmídeo <ol><li> Gerar os plasmídeos de ADN para electroporação in vivo por subclonagem do ADNc ou sequência de shRNA em um plasmídeo de expressão para células de mamífero 12. Usar um citomegalovírus precoce imediato intensificadoras e de frango β-actina promotor de fusão (CAG) plasmídeo conduzido pelo promotor 13 porque permite a expressão forte e estável. Para a expressão de shRNAs sob o controlo de um promotor CAG, usar um sistema de cassete shRNA baseada mir30 para subclonagem a 14 shRNA. </li><li> Purifica-se DNA de plasmídeo com um kit Maxi-prep de acordo com as instruções do fabricante, e ressuspender o ADN com solução salina tamponada com HEPES (NaCl 140 mM, 0,75 mM de Na 2 HPO 4, 25 mM de HEPES, pH 7,40). Ajustar a concentração de ADN para ≥4 ug / uL. </li><li&gt; Preparar a solução de DNA de plasmídeo a uma concentração de 4 mg / mL, e adicionar uma quantidade mínima de corante verde rápido (concentração final de 0,01%) para marcar o local da injecção. Quando é necessária a electroporação simultânea de vários plasmídeos, ajustar a concentração total da solução de DNA de plasmídeo a 4 mg / mL. Nota: A composição óptima dos DNAs de plasmídeo deve ser determinado de acordo com a eficiência de transfecção de cada plasmídeo. </li></ol> 2. A esterilização de instrumentos cirúrgicos e Saline <ol><li> instrumentos cirúrgicos autoclave e a solução de NaCl a 0,9%. </li></ol> 3. Preparação da micropipeta de vidro <ol><li> Puxe pipetas de vidro usando um extrator pipeta. Cortar a ponta da pipeta para um diâmetro de 30-50 ^ m. Utilize os seguintes parâmetros: calor, 600; Velocidade, 50; Tempo, 75. </li></ol> 4. Cirurgia animal, Injecção de ADN e electroporação Nota: Um excessoFace a esta etapa é descrita na Figura 1. Embora o procedimento de crias de ratos são descritos aqui, o método também é aplicável a animais mais maduros, utilizando o mesmo procedimento. <ol><li> Anestesiar ratos com isoflurano na caixa de indução até que o animal torna-se imóvel, ajustando o fluxo de oxigénio para 0,4 L / min e a concentração de isoflurano a 4% (vol / vol). Execute toe beliscar para confirmar anesthetization adequada. </li><li> Colocar o rato sobre o aquecedor pré-aquecido sob um microscópio binocular e manter a anestesia por administração de isoflurano continuamente através da máscara facial. Ajustar o fluxo de oxigénio para 0,2 L / min e a concentração de isoflurano a 2% (vol / vol). Use colírios para evitar a secura dos olhos, se os olhos do animal estão abertas. </li><li> Fixar as pernas com fita cirúrgica. </li><li> Limpar a pele na parte posterior da coxa com povidona-iodo, e fazer uma incisão com um bisturi. Nota: Raspar áreas cirúrgicas se as áreas cirúrgicas são cobertas com har. </li><li> Expor o nervo ciático, criando uma abertura entre os músculo quadríceps femoral e músculo bíceps femoral com agulhas de costura. </li><li> Molhar o nervo com uma solução de NaCl a 0,9%. Absorver o excesso de água com papel sem fiapos. </li><li> Inserir a base de uma micropipeta de vidro na tubagem flexível, e preencher a quantidade adequada de solução de ADN (pelo menos um microlitro) na micropipeta, aspirando suavemente. </li><li> Levantar o nervo exposto puxando suavemente o lado distal do nervo usando uma agulha. Nota: Não aplicar tensão no nervo para minimizar o stress mecânico. </li><li> Inserir a micropipeta de vidro no local distai do nervo, e injectar a solução de DNA através da aplicação de pressão (isto é, por sopro para dentro da extremidade aberta do tubo flexível). Injectar a solução de DNA até o nervo aparece verde (máximo de 1 ml). Porque inserção frequente da micropipeta pode danificar o nervo, não insira o micropipeta mais de duas vezes. </li><li> Coloque uma TWEezer tipo eletrodo de platina cerca de 1-2 mm do nervo. Preencher a lacuna entre o eletrodo e o nervo com uma solução de NaCl a 0,9%. Nota: Não segure o nervo com o eletrodo para evitar estresse mecânico sobre o nervo. </li><li> Aplicar impulsos eléctricos para o local da injecção usando um electroporador com o eléctrodo. Após o primeiro conjunto de pulso, inverter o eletrodo e aplicar outro conjunto de pulso. Utilize os seguintes parâmetros: tensão, 50 V; duração do pulso, 5 ms; intervalo de pulso, a 100 ms; número de pulsos, 4 vezes. </li><li> Limpar o local de electroporação com uma solução de NaCl a 0,9%. </li><li> Repita os passos 4,4-4,11 sobre o nervo ciático contralateral. </li></ol> 5. Pós-eletroporação <ol><li> Fechar as incisões com cola de cianoacrilato. </li><li> Após a secagem da cola, limpar a ferida com povidona-iodo. </li><li> Solte o filhote de cachorro da máscara facial. Aqueça o filhote em um mais quente, pelo menos durante uma hora, a fim de permitir que ele se recuperar totalmente da anestesia. Do não deixar o cachorro sem supervisão até que tenha recuperado a consciência suficiente. </li><li> Após a recuperação da anestesia, devolver o cachorro para o rato mãe. Não devolva o filhote até que esteja totalmente recuperado. </li></ol> 6. Pós-cirurgia <ol><li> Casa os filhotes de ratos na gaiola até a realização dos experimentos 11 (ver exemplos na Figura 3). Administrar carprofeno (5 mg / kg; ip), uma droga não-esteróide anti-inflamatório, ou buprenorfina (0,1 mg / kg, sc), um analgésico opióide, se necessário. Nota: Se o filhote de rato não cresce bem ou inflamação é observado ao redor do local da cirurgia, excluir o animal a partir dos experimentos. </li></ol>

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The authors declare that they have no competing financial interests.

In this paper, we describe a simple and efficient method that allows in vivo gene transfer to myelinating SCs in the rat sciatic nerve using electroporation. This method allows highly selective gene expression in myelinating SCs by simply applying electric pulses to the plasmid DNA-injected sciatic nerve. Because the molecular mechanisms of myelination and demyelination in the peripheral nervous system remain unclear, the present in vivo electroporation method will be a powerful tool to clarify the roles of multiple genes of interest in living animals.
A critical requirement of this method is to keep damage to the nerve during surgery to a minimal level. Should surgical damage cause excessive inflammation, the sciatic nerve may degenerate. To avoid this, one must conduct surgery with extreme care, so as to not damage the blood vessels around the nerve. Mechanical stress to the nerve during the surgery can also be a cause of nerve damage. To minimize mechanical stress, lifting the exposed nerve should be done as gently as possible, and the tweezer-type electrode should be placed close to the nerve without contact. Furthermore, electrical pulses that are too strong can cause undesirable large leg movement, which leads to mechanical stress, or can burn the nerve. If significant damages are observed in the nerves, we recommend reducing the electrical pulse intensities or placing the electrode further away from the nerve.
In our present protocol, CAG promoter-driven plasmids were used as expression vectors. CAG promoter-driven plasmids allow high levels of gene expression in myelinating SCs in vivo. We also have tried a CMV promoter, another widely used universal promoter for mammalian gene expression, but expression of the gene product was very weak. This is consistent with previous results, in which electroporation-mediated transfection was conducted in the embryonic brain20. Therefore, we recommend using CAG promoter-driven plasmids for the in vivo electroporation method.
Because axonal signaling is a key factor in myelination/demyelination21, gene modification in neurons is also important. However, delivery of transgenes using our in vivo electroporation method is limited to SCs. It has been reported that gene delivery into sciatic nerve axons can be achieved when in vivo electroporation is applied to dorsal root ganglion (DRG) neurons in adult rats22. This suggests that delivery of plasmid DNA to the cell body is likely to be critical for in vivo transfection of peripheral axons. Thus, to examine the involvement of axonal molecules in myelination/demyelination, researchers should use neuron-specific genetic methods such as genetically modified animals, neuron-specific viral vectors, or in vivo electroporation to DRG neurons.
Compared with current methods, such as the generation of genetically modified animal lines23 and delivery of transgenes by viral vectors4-6, gene modification of SCs by in vivo electroporation is simpler. This method only requires several days for plasmid DNA construction and one day for electroporation surgery. Plasmid DNA construction does not require a biohazard room that is usually essential for viral vector handling. In addition, one of the advantages of the electroporation method is the capacity for simultaneous expression of multiple gene products using a simple protocol. Our novel technique will be useful for analyzing the interaction of a variety of signaling molecules involved in myelination and demyelination. In particular, by permitting the cotransfection of a number of different intracellular fluorescent probes, our method should be a powerful tool for investigating intracellular signaling dynamics in SCs using live imaging experiments.

The rapid transmission of sensory and motor information in the peripheral nervous system is permitted by the myelin sheath, which is formed by myelinating Schwann cells (SCs)1. Insulation of axons by the myelin sheath enables saltatory conduction, which increases the speed of nerve impulses. In disorders in which the development or maintenance of the myelin sheath is impaired, nerve conduction speed is reduced. This results in neuropathy involving motor and sensory dysfunction. Although there are many studies on the molecular mechanisms of myelination and demyelination in the peripheral nervous system, the roles of the numerous proteins involved in these processes remain unclear.
To study the molecular mechanisms of SC myelination/demyelination in vivo, genetic approaches have been used to modify gene expression in animals. A powerful approach is the use of knockout/knockin or transgenic animals. However, the generation of these animals is expensive and time consuming. For SC-specific gene manipulation, crossing floxed strains with Cre mice or other conditional gene expression methods are necessary. This again is laborious and time intensive. In recent years, a cutting-edge genetic technology, the CRISPR-Cas9 system, has made the generation of genetically modified mice much quicker (about 4 weeks)2,3, but this method is hindered by target sequence limitations, and suffers from off-target effects. As an alternative method, viral vector-mediated gene transfer is a faster and easier method of achieving gene transfer into SCs in vivo4-6. Indeed, the generation of viral vectors is less expensive, and takes a shorter time (within a few weeks), and gene manipulation of SCs can be achieved by simply injecting engineered viral vectors, such as adenoviral vectors, adeno-associated viral (AAV) vectors, and lentiviral vectors, into the sciatic nerve. Because these viral vectors have different characteristics, users have to choose the one best suited for their purpose. Adenoviral vectors infect axons and SCs in both young and mature sciatic nerves. In particular, adenoviral vectors have higher selectively for non-myelinating SCs than myelinating SCs. Adenoviruses can cause immune responses, and accordingly, immunodeficient strains should be used5. AAV vectors are currently the most widely used viral vectors, and allow in vivo gene transfer with lower toxicity7. AAV can transduce both axons and SCs by direct injection into the nerve fibers8,9. However, AAV-mediated protein expression usually requires 3 weeks or longer to reach maximum levels7,9. Therefore, it is difficult to analyze myelination, which actively progresses during the two week postnatal period. Lentiviral vectors have higher selectively for myelinating SCs than non-myelinating SCs, and do not have toxic effects on sciatic nerves. However, lentiviral vectors do not infect SCs in more mature nerves5, and therefore are unsuitable for analyzing events such as the demyelination process.
Electroporation is another faster and easier approach to achieve in vivo gene transfer. It has been reported that in vivo transfection of SCs can be achieved when electroporation is applied to transected rat sciatic nerves10. However, because this method requires nerve transection for gene delivery, the application is limited to the analysis of the damaged nerves. Here, we describe an alternative method that allows the delivery of transgenes into myelinating SCs in intact rat sciatic nerves using electroporation11. This method requires plasmid construction, which can usually be completed within a week. Then, by simply delivering electric pulses to the site on the sciatic nerve where the plasmid DNA was injected, highly selective transfection of myelinating SCs can be achieved in neonatal as well as in more mature animals. By electroporating multiple plasmids, simultaneous expression of a variety of genes can be easily achieved. The ability to simultaneous express multiple molecules, such as signaling proteins, short-hairpin RNAs (shRNAs) and functional probes, is crucial for investigating complex processes such as myelination and demyelination. The novel in vivo electroporation method described in this paper will be a powerful tool, allowing researchers to analyze the function of a multitude of molecules and their interactions in myelinating SCs.

<table><tbody><tr><td>Genopure Plasmid Maxi Kit</td><td>Roche</td><td>03 143 422 001</td><td>Plasmid DNA purification kit</td></tr><tr><td>Fast Green CFC</td><td>WAKO</td><td>069-00032</td><td>Dye for DNA injection</td></tr><tr><td>GC 150T-10</td><td>HARVARD APPARATUS</td><td>30-0062</td><td>Glass capillary</td></tr><tr><td>Suction tubing</td><td>Drummond</td><td>05-2000-00</td><td>Suction tubing for micro injection</td></tr><tr><td>MODEL P-97</td><td>SUTTER INSTRUMENT CO.</td><td>Micropipette puller</td><td></td></tr><tr><td>CUY21 Single Cell</td><td>BEX</td><td>Electroporator CUY21 Single Cell</td><td>Pulse generator</td></tr><tr><td>Electric warmer</td><td>KODEN</td><td>CAH-6A</td><td>Warmer during the surgery</td></tr><tr><td>Isofluolane</td><td>Mylan</td><td>1119701G1076</td><td>Anesthetic</td></tr></tbody></table>

in vivo gene transfer to schwann cells in the rodent sciatic nerve by electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

Um exemplo de um nervo ciático transfectada com proteína fluorescente vermelha (RFP) -expressing plasmídeo é mostrada na Figura 2A. As células que mostram morfologia bipolar, uma característica de SCs, foram escassamente transfectadas com RFP. Nenhuma fluorescência RFP foi detectado em axónios. Normalmente encontramos ~ 100 SCs transfectadas em cada nervo. Esta eficiência de transfecção parece semelhante à in vivo SC eficiência de infecção utilizando vectores lentivirais 4. Experimentos imunocoloração mostrou que a maioria (~ 96%) as células RFP-positivo em P7 co-marcadas durante S100, um marcador SC (Figura 2B), e 91% de células RFP-positivo em P14 co-marcadas durante MBP, um marcador mielinizantes SC (Figura 2C), o que sugere que a transferência genética através de electroporação é altamente selectivo para myelinating SCs. A introdução de genes em múltiplas SCs <em&gt; in vivo será extremamente útil para investigar os mecanismos de mielinização / desmielinização. Uma grande vantagem do método de electroporação in vivo descrito aqui é a capacidade de transferência de genes múltiplos com um procedimento simples. A Figura 2D mostra uma imagem representativa de um nervo ciático transfectadas com uma mistura de GFP e os plasmídeos que expressam RFP utilizando electroporação em vivo. Cerca de 97% das SC foram GFP e RFP duplo positivo, sugerindo que a entrega altamente eficiente de genes múltiplos pode ser conseguido simplesmente electroporating misturas de vários plasmídeos. Em roedores, a mielinização inicia em torno do nascimento, aumentando dramaticamente durante as primeiras duas semanas após o parto, e depois diminui gradualmente. Assim, manipulando geneticamente SCs durante essas janelas de tempo de desenvolvimento, os mecanismos subjacentes a estas diferentes fases de mielinização pode ser esclarecida. lentivirais são uma boa ferramenta para umnalyzing mielinização, especialmente como eles têm uma toxicidade mínima, mas lentivírus única infectar neonatal nervo ciático 5,6. Em comparação, a transferência de genes mediada por electroporação funciona bem quando a transfecção é realizada na P3 (Figura 2E, topo) ou em P14 (Figura 2E, parte inferior). As aplicações do novo método em electroporação in vivo são descritos aqui. A Figura 3A mostra que as imagens ao microscópio óptico de GFP-expressando myelinating SCs em várias fases do desenvolvimento (P7, P14, P21 e P31). Por análise microscópica de luz, alterações nos parâmetros morfológicos, tais como comprimento e diâmetro, pode ser avaliado. Note-se que estes parâmetros têm valores semelhantes em comparação com ratos intactos 15,16 nervos periféricos, o que sugere que os nervos electroporadas desenvolver, sem efeitos prejudiciais significativos. A Figura 3B mostra uma imagem microscópica de electrões de LacZ-expressing myelinating SCs. Neste caso, LacZ foi utilizada como um marcador de expressão. β-galactosidase coloração utilizando Bluo-gal, um substrato insolúvel em etanol, possibilita a análise da estrutura de mielina de SCs transfectadas por microscopia eletrônica 11,17. Nestas experiências, o papel de moléculas de sinalização pode ser examinado por silenciamento ou aumentando a sua expressão, permitindo assim a análise da perda de função ou efeitos de ganho de função. Para além da análise de tecido fixo, in vivo, transferência de genes mediada por electroporação pode ser também aplicado a viver experiências de imagiologia. Por exemplo, a Figura 3C mostra uma mielinizantes SC co-expressando G-GECO1.1 18, um indicador verde fluorescente citosólica de Ca2 +, e R-GECO1mt 19, um indicador vermelho fluorescente Ca2 + mitocondrial. Ao expressar estes indicadores, identificamos uma via de sinalização que controla citosólica e mitocondrial de Ca2 + concentrações em myelinating SCs<html> . Assim, o presente método pode ser usado para estudar uma variedade de mecanismos de sinalização, especialmente quando as sondas fluorescentes geneticamente codificados estão disponíveis para detectar os sinais de interesse. <img alt="figura 1" src="/files/ftp_upload/54567/54567fig1.jpg" /> Figura 1:. Esquemática do i n vivo A electroporação Método Em primeiro lugar, o nervo ciático do rato anestesiado é exposta. Em segundo lugar, o ADN de plasmídeo é injectado no nervo ciático. Terceiro, os impulsos eléctricos são entregues no local da injecção, através do eléctrodo de uma pinça em forma de. Finalmente, a ferida é fechada com cola. Este procedimento pode ser repetido sobre o nervo contralateral. <a href="https://www.jove.com/files/ftp_upload/54567/54567fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> &quot;&gt; <img alt="Figura 2" src="/files/ftp_upload/54567/54567fig2.jpg" /> Figura 2: Resultados representativos sobre nervos ciáticos transfectadas (A) uma imagem representativa de um nervo ciático transfectadas.. O nervo foi transfectada com o plasmídeo que expressa RFP no P3, e fixado em P7. (B) uma imagem representativa de uma célula transfectada-RFP em P7 mostrando uma co-localização com S100, um marcador SC. (C) uma imagem representativa de um nervo ciático transfectada-RFP no P14 mostrando uma co-localização com MBP, um marcador mielinizantes SC. (D) A imagem representativa de um nervo ciático cotransfectadas com GFP e plasmídeos que expressam RFP. SCs transfectadas expressaram simultaneamente GFP e RFP. (E) Uma imagem de myelinating SCs no P31 transfectadas em P3, quando mielinização começa (em cima), e uma imagem de SCs myelinating no P31 transfectadas a P14, quando a maioria das grandes axônios se tornar mielinizado (parte inferior), sugerindo que a transfec ç ã o das SC myelinating pode ser conseguida não só em nervos neonatal, mas também em nervos mais maduros. Barras de escala = 200 uM (A); 50 mm (BE). Este valor foi modificado a partir de nossa publicação anterior 11. <a href="https://www.jove.com/files/ftp_upload/54567/54567fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54567/54567fig3.jpg" /> Figura 3: Aplicação de electroporação in vivo (a) uma análise microscópica de luz do desenvolvimento de myelinating SCs.. Os nervos ciáticos foram electroporadas com o plasmídeo que expressa GFP em P3, e foram fixados em vários estágios de desenvolvimento (P7, P14, P21 e P31). Imagens representativas de CT-GFP positivo estão apresentados à esquerda. A duração média e o diâmetro estão resumidos como média ± EPM (n = 30-47 de 3 nervos) no lado direito. O comprimento e diâmetro do myelinating SCs aumenta à medida que prossegue o desenvolvimento. (B) uma imagem de microscopia electrónica de um nervo ciático transfectadas com um plasmídeo que codifica para LacZ. Um transf SC (asterisco branco, à esquerda) foi finamente marcado com precipitados do produto da reacção de β-galactosidase. (C) Uma imagem de um SC cotransfectadas com G-GECO1.1, um indicador verde fluorescente citosólica de Ca2 +, e R-GECO1mt, um indicador vermelho fluorescente mitocondrial de Ca2 +. As regiões dentro dos retângulos pontilhadas brancas são mostrados ampliada nos painéis à direita. Barras de escala = 50 uM (A e C); 1 | iM (B). Este valor foi modificado a partir de nossa publicação anterior 11. <a href="https://www.jove.com/files/ftp_upload/54567/54567fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation at JoVE.com

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Em Gene Vivo Transferência de células de Schwann no nervo ciático de roedores por Eletroporação

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

in-vivo-gene-transfer-to-schwann-cells-rodent-sciatic-nerve

Research

JoVE Journal

Neuroscience

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

8.9K visualizações.  The University of Tokyo. Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Vídeo: In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Transfecção de células de camundongo por Ganglionares da Retina In vivo Eletroporação

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Neurociência

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Em Eletroporação vivo de Retina do rato em desenvolvimento

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

Entrega Gene eficiente em múltiplos territórios CNS Usando In Utero Eletroporação

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

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 ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Em Medidas Vivo eletrofisiológicos nos nervos ciático Rato

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Subtipo seletivo Eletroporação de Cortical Interneurônios

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord injury. The complete transection model, although not as clinically relevant as the contusion model, is the most rigorous method to evaluate axon regeneration. In the contusion model, it is difficult to distinguish regenerated from sprouted or spared axons due to the presence of remaining tissue post injury. In the complete transection model, a bridging method is necessary to fill the gap and create continuity from the rostral to the caudal stumps in order to evaluate the effectiveness of the treatments. A reliable bridging surgery is essential to test outcome measures by reducing the variability due to the surgical method. The protocols described here are used to prepare Schwann cells (SCs) and conduits prior to transplantation, complete transection of the spinal cord at thoracic level 8 (T8), insert the conduit, and transplant SCs into the conduit. This approach also uses in situ gelling of an injectable basement membrane matrix with SC transplantation that allows improved axon growth across the rostral and caudal interfaces with the host tissue.

This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.

Transplante de Schwann células dentro de conduítes de PVDF-TrFE a ponte rato necrosante tocos de medula espinhal para promover a regeneração do axônio através da abertura

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord ...

Medicina

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Investigação regeneração do axônio mamíferos: In Vivo Electroporation do gânglio de raiz Dorsal do rato adulto

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

In Vivo SiRNA transfecção e Gene Knockdown em Spinal Cord via rápida não invasiva lombar intratecal Injeções em Ratos

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Biologia

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

Mielinização in vitro de axônios periféricos em cocultura de explantes de gânglios dorsais de rato e células de Schwann

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

Em Gene Vivo Transferência de células de Schwann no nervo ciático de roedores por Eletroporação

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