Este trabalho foi financiado pela Fundação Alemã de Pesquisa (Deutsche Forschungsgemeinschaft).

1. Preparação de DNA e vidro Micropipetas para Injeção <ol><li> Bons micropipetas de vidro de qualidade é essencial para reduzir a taxa de abortos inicial elevado, devido à perda de líquido amniótico. O procedimento para puxar micropipetas de vidro tem sido bem documentada 13,18,25. Use 1,2 milímetros de diâmetro capilares puxado em um dispositivo convencional Sutter P-97 com as configurações de P = 500; Calor = 300; Puxe = 40; Velocity = 50; Time = 50. Monte o extrator com três milímetros filamentos &quot;vale&quot; (Sutter Instrument FT330B). Os filamentos de dois milímetros de tamanho produziram para nós, resultados menos satisfatórios. Por outro lado, o bisel da micropipeta dicas não parece melhorar os resultados para nós. </li><li> Dissolver purificada, isenta de endotoxinas DNA de plasmídeo em PBS (Grau cultura celular) contendo 0,1% de verde rápido para uma concentração final de 1 a 2 mg / mL. O verde rápido tornará visível a solução injectada no ventrículo do cérebro embrionário. </li><li> Carregar 10 ml de solução de ADN para a micropipeta de vidro. </li&gt; <li> Ligue a micropipeta de vidro para o sistema de injeção (bomba Pico) ou pipeta de boca. </li></ol> 2. Anestesia Preparar a mesa cirúrgica com uma almofada de aquecimento e os instrumentos cirúrgicos. Ligar as fontes de luz fria para facilitar a visualização dos embriões. Desinfetar os instrumentos cirúrgicos, utilizando, por exemplo, um talão esterilizador de vidro. Três procedimentos de anestesia diferentes são possíveis para este protocolo (ver Discussão). Aqui descrevemos a que consideramos o melhor, usando inalação de isoflurano para a indução da anestesia (taxa de fluxo de 0,5 L / min), bem como a manutenção (caudal de 1 L / min). <ol><li> Introduzir o mouse grávida em uma pequena transparente (para que o rato permanece visível) recipiente ligado ao Marcos 5 anestésico dispositivo Komesaroff por um curto período de tubulação. </li><li> Encher o vaporizador com isoflurano, em seguida, abrir o frasco de oxigénio ligado ao dispositivo. Soprar um ou dois pulsos de isoflurane / oxigênio (0,5 L / min) para o recipiente com o mouse e relógio para narcose para definir pol </li><li> Leve o mouse para fora, cubra seus olhos com pomada para impedi-los de secagem e ajustar a máscara de anestesia o fluxo em sua cabeça imediatamente. </li></ol> 3. Laparotomia <ol><li> Posicione o mouse de barriga para cima sobre a almofada de aquecimento (caso contrário, a temperatura do corpo vai descer muito rápido, diminuindo as chances de recuperação) e garantir o seu corpo no lugar, fixando suas quatro pernas para os lados com fita adesiva. Avaliar a profundidade da anestesia através da verificação de perda de resposta à estimulação de reflexo (por exemplo, do dedo do pé ou de aperto da cauda, ​​com uma pressão firme). </li><li> Raspar a superfície abdominal e desinfectá-la com o iodophorpovidone-iodo (Braunoderm). </li><li> Adicione uma incisão longitudinal (1 a 1,5 cm de comprimento) sobre a pele abdominal. Em seguida, corte o peritônio. Coloque gaze de algodão ao redor da incisão. Faça um corno uterino visível e puxe-o cuidadosamente com uma pinça sem corte para o PBS-lavadas gauze. Lavar o útero com PBS muito frequentemente para mantê-lo sempre humedecido (Figuras 2A e 2B). Durante todo o procedimento evitar puxar o mesométrio ou do útero apertado, uma vez que a alta pressão no interior do útero irá transmitir ao embrião aumentando as chances de perda de líquido sobre injeção, resultando em aborto. </li></ol> 4. Injecção de DNA para o ventrículo cerebral <ol><li> Segurar o útero de tal forma que os ventrículos cerebrais podem ser visualizados. Não extensivamente reposicionar um embrião que se encontra em uma posição desfavorável - isso só aumenta as chances de aborto. </li><li> Olhando para a cabeça do embrião a partir de cima, localizar visualmente o espaço ou fissura entre os hemisférios esquerdo e direito do córtex. Os hemisférios são fáceis de distinguir e os ventrículos laterais dentro deles (a não ser alvo) pode geralmente ser entendido como formas ligeiramente mais escuras. Se um embrião é encontrado para ser perfeitamente orientada para a injeção de DNA (Figures 2C e 2D), é possível perfurar a parede uterina e se introduzir o terceiro ventrículo de uma vez. Segurar a micropipeta de vidro a 45 ° em relação à parede do útero e punção no final rostral do fosso entre os hemisférios corticais, penetrando para cerca de 1 mm. Deste modo, a ponta da micropipeta de vidro vai entrar no terceiro ventrículo do cérebro (não no ventrículo lateral) (Figuras 2C e 2D). Em embriões menos favorável orientadas é útil para perfurar a primeira parede uterina (sempre a 45 °), na vizinhança da cabeça embrionária, colocar a ponta da micropipeta na posição correcta entre os hemisférios corticais e só depois perfurar o cérebro. Injectar cerca de 1 ul de solução de ADN para o terceiro ventrículo (uma boa injecção enche com fluido no ventrículo verde). Repita o mesmo procedimento com todos os embriões de um chifre útero. Isso permite algum tempo para a solução de ADN para misturar uniformemente com o fluido atingir o ventricular e entire neuroepithelium. </li></ol> 5. Segmentação do hipotálamo para Eletroporação <ol><li> Desligue o electroporator e ajustar as configurações de acordo com a idade embrionária (para E12.5 usamos 5 pulsos de onda quadrada, 50 V, 50 ms ON/950 ms OFF). Use como pólo positivo do eletrodo de agulha de aço inoxidável (CUY550-10) e como pólo negativo de um eletrodo liso rodada (CUY700P4L). (É importante que os eléctrodos são menores do que o embrião, pois caso contrário, a corrente flui através do embrião, mas não por ele.) </li><li> Selecione para eletroporação os embriões cujo lado dorsal é para cima (ou seja, voltada para o experimentador) (como a maioria deles são), e descartar aqueles cuja orientação não é favorável. Agora é seguro para tocar o útero com os dedos etanol desinfetadas ou com luvas. Segurar o útero com uma pinça, no entanto, iriam interferir com o fluxo de corrente durante a electroporação. </li><li> Perfuram a parede uterina por cabeça do embrião entre a amnsac iotic ea placenta empurrando para baixo com ele através da ponta do eléctrodo de agulha. Use o dedo indicador da outra mão, como bloco de empuxo. Cerca de 5 mm da ponta do eléctrodo deve ser agora entre o saco amniótico e a parede uterina, a cerca do nível do mesencéfalo (figuras 2E e 2F). Lembre-se que este é o &quot;alvo eletrodo&quot;, pelo que a sua posição vai determinar qual parte do neuroepithelium hipotálamo é o mais provável de ser transfectadas. O embrião tem que ter muito cuidado &quot;espremido&quot; entre os eletrodos. </li><li> Com a outra mão, a posição do eléctrodo plano rodada do lado de fora da parede do útero, no lado oposto da cabeça do embrião (Figuras 2E e 2G). </li><li> Utilize o pedal para aplicar tensão (50 V, 50 ms ON, 950 ms OFF, 5 pulsos de onda quadrada). </li><li> Puxe lentamente o eletrodo de agulha para fora do útero, mantendo o útero de volta com o dedo indicador da outra mão - se amniocentesetic líquido é perdida através da parede perfurada, usualmente o embrião vai sofrer aborto. Repita o procedimento com todos os embriões. </li><li> Voltar trompa do útero no abdômen e repetir a injeção e eletroporação no corno restante. </li></ol> 6. Finalizando a cirurgia <ol><li> Após a injeção e eletroporação de todos os embriões, coloque o útero de volta ao abdômen com muito cuidado, posicionado exatamente como eram antes e úmido da cavidade peritoneal com solução salina antes de fechá-la. </li><li> Sutura do peritônio com Categutes (Vicryl poliglactina 910, 5-0, Ethicon V4914). Para o encerramento do uso de pele &quot;, interrompeu ponto&quot; methodwith uma sutura mais resistente (Supramid Nylon, 6-0, Serag Wiessner TO 07171L). </li><li> Desinfectar a superfície abdominal com iodo-povidona (Braunoderm) e injectar um não-esteróide anti-inflamatório, por via subcutânea (por exemplo, 100 ul de uma solução a 1:10 de Rimadyl em 0,9% de NaCl), ou, ainda melhor, um opiáceo como buprenorphine (Temgesic, 0,05 a 0,1 mg / kg de peso corporal em 0,9% de NaCl), para aliviar a dor. </li><li> Remover a mãe do aparelho de anestesia e colocá-lo em uma gaiola, que é aquecida por uma almofada de aquecimento. Monitorizar o rato continuamente até que esteja completamente recuperado da anestesia. Mais tarde, verifique os animais diariamente para garantir que eles estão se recuperando do procedimento, sem qualquer sinal de infecção ou dor. </li></ol> 7. Analisando os resultados <ol><li> Colher os embriões (ou postnatals) de acordo com o dia da análise desejada. Ratos pós-natais (1 a 2 dias de idade) são sacrificados por decapitação. </li><li> Separar cada embrião de acordo com a anotação electroporação anterior. Dissecar o cérebro sob um estereomicroscópio e vá ao microscópio para o sinal fluorescente verde (GFP a partir do repórter) na região adequada. </li><li> O cérebro seleccionado pode ser analisada &quot;fresco&quot;: fixar o tecido durante um curto período de tempo, em 4% de paraformaldeído e incorporar em agarose a 4% e em microesferas de gelatina a umlbumin, em seguida, a secção de um dispositivo do tipo vibratome (Figura 3). Para uma análise mais pormenorizada imunohistoquímica (Figura 4), ​​crio-protegem o cérebro em solução de sacarose a 30%, em outubro incorporar o meio de montagem (Tissue Tek) depois cortar secções 20 mM num criostato. </li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+silencing+of+Sry+gene+with+RNA+interference+in+developing+mouse+embryos+resulted+in+feminization+of+XY+gonad.">Effective silencing of Sry gene with RNA interference in developing mouse embryos resulted in feminization of XY gonad.</a> J. Biomed. Biotechnol. 2012, 343891 (2012).</li><li>Kikuchi, N., Nakamura, S., Ohtsuka, M., Kimura, M., Sato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+mechanism+of+gene+transfer+into+early+to+mid-gestational+mouse+fetuses+by+tail+vein+injection.">Possible mechanism of gene transfer into early to mid-gestational mouse fetuses by tail vein injection.</a> Gene Ther. 9, 1529-1541 (2002).</li><li>Foust, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat. Biotechnol. 27, 59-65 (2009).</li><li>Xin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+targeting+mechanism+of+Angiopep-conjugated+poly(ethylene+glycol)-co-poly(epsilon-caprolactone)+nanoparticles.">The brain targeting mechanism of Angiopep-conjugated poly(ethylene glycol)-co-poly(epsilon-caprolactone) nanoparticles.</a> Biomaterials. 33, 1673-1681 (2012).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108, 193-199 (1991).</li><li>Kaufman, R. J., Davies, M. V., Wasley, L. C., Michnick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+vectors+for+stable+expression+of+foreign+genes+in+mammalian+cells+by+use+of+the+untranslated+leader+sequence+from+EMC+virus.">Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.</a> Nucleic Acids Res. 19, 4485-4490 (1991).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu. Rev. Biochem. 67, 509-544 (1998).</li><li>Londrigan, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+promoters+for+driving+efficient+transgene+expression+in+neonatal+porcine+islets.">Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets.</a> Xenotransplantation. 14, 119-125 (2007).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Sobre a anestesia: Como no útero electroporação no hipotálamo pode ser tecnicamente difícil e narcose requerem tempos mais longos, preferimos para induzir e manter a anestesia por meio da administração de uma mistura de oxigénio e isoflurano. Na nossa experiência, os animais podem permanecer convenientemente anestesiados deste modo por períodos de até, pelo menos, uma hora, a recuperação da mãe é muito rápido, e melhorou a sobrevivência dos embriões. Outras abordagens para a anestesia, também...

A manipulação genética do cérebro embrionário do rato é uma abordagem preferida para aprender sobre regulação do desenvolvimento. A geração de linhas mutantes de rato, porém, é lento e caro. Um método poderoso para introduzir alterações genéticas específicas no desenvolvimento de neurónios do cérebro de mamíferos está em electroporação útero. Essencialmente, a técnica consiste na transfecção de DNA para o neuroepitélio cérebro embrionário, por meio de impulsos eléctricos, em seguida, permitindo que o embrião para sobreviver durante um certo período de tempo, recolher o cérebro e examiná-las para possível nova, fenótipos informativos. Desta forma, o experimentador pode testar hipóteses quase imediatamente, sem longos períodos de espera necessários para a produção de mutantes de rato. Transfecção de DNA em embriões em desenvolvimento começou com a eletroporação in ovo em embriões de galinha 1. A prova-de-conceito essencial para o rato foi realizado na cultura <sup&gt; 2. Isso foi logo seguido pelas primeiras descrições da técnica no mouse no útero 3,4. O principal problema é a transfecção do cérebro em desenvolvimento de embriões no útero, sem as matar ou a mãe. Aprender a realizar a cirurgia necessária (laparotomia, injeção, eletroporação) exige um longo período de treinamento. Uma vez que a cirurgia foi dominado até o ponto onde a taxa de sobrevivência do embrião é aceitável, a próxima pergunta chave é: quais estruturas cerebrais são acessíveis? Não surpreendentemente, os primeiros trabalhos publicados que mostram os resultados obtidos com a eletroporação utero focada no desenvolvimento cortical 5-9. O mesmo é válido para a maioria das publicações utilizando esta técnica, uma vez que a região do cérebro do rato em desenvolvimento mais acessíveis para procedimentos cirúrgicos é o córtex (Figura 1). O procedimento para a electroporação útero no córtex foi descritoem versão impressa e 10 em vídeo 11-14. Uma modificação da técnica pode ser usada para atingir uma parte ventral do telencéfalo, os gânglios da base 15. Além do telencéfalo, diencéfalo (classicamente dividido em tálamo e hipotálamo) é uma região do cérebro anterior mais difícil de alcançar. Um pequeno número de relatórios de trabalhos de segmentação de sua parte dorsal e mais acessível, o tálamo 16-19. O hipotálamo é a parte mais ventral do cérebro anterior, por conseguinte, a uma localizada mais profundamente da face dorsal (córtex) (Figura 1). Esta região continua a ser um desafio difícil para os pesquisadores comprometidos com a manipular geneticamente o cérebro do rato no útero. Para nosso conhecimento, só muito poucos artigos relatam os resultados de transfecção in utero no hipotálamo do rato 20,21. No entanto, a importância funcional do hipotálamo Canhãot ser exagerada, uma vez que regula comportamentos como comer e beber, acasalamento, reprodução e parentalidade 22. Além disso, alterações no desenvolvimento do hipotálamo contribuir para originar mais tarde, em condições de vida, como obesidade, hipertensão, diabetes e puberdade precoce 23. Ser capaz de alterar geneticamente o hipotálamo durante o desenvolvimento seria uma ferramenta muito poderosa para compreendê-lo. O protocolo cirúrgico de base para a laparotomia de ratas grávidas que usamos aqui é similar ao utilizado em outros protocolos 11,13,14,24. Vamos descrevê-los aqui brevemente para ser completo. A chave para o nosso processo, por outro lado, são do tipo de anestesia, o local de injecção, do tipo de eléctrodos e a posição de inserção e o eléctrodo positivo em relação à cabeça do embrião. Nós preferimos para induzir e manter a anestesia por inalação de gás sobre anestesia intraperitoneal simples, uma vez que a primeira permite que peloum pouco mais longos períodos de narcose necessária para uma cirurgia difícil. Isoflurano resultados inalação em uma recuperação mais rápida da anestesia, já que geralmente a mãe demonstra um comportamento normal já minutos após a cirurgia. O ponto de injecção mais fácil da solução de ADN com a micropipeta de vidro é o ventrículo lateral, que, contudo, é totalmente inadequado para electroporação hipotálamo. Injeção direta no terceiro ventrículo é de fato crucial para atingir estruturas diencefálica profundas. É possível transfecção do hipotálamo ou da E12.0 E12.5 com eletrodos padrão, off-the-shelf. Encontraram-se alguns dos eléctrodos fabricados por Nepa Gene (Chiba, Japão), particularmente adequado para esta finalidade. Com o nosso procedimento obtemos transfecção de todo o neuroepitélio hipotalâmica ou transfecção parcial, dependendo da orientação da região do eléctrodo. Aqui vamos demonstrar a técnica de transfecção do corpo mamilar, sem dúvida,o mais profundo e recesso de todos os núcleos do hipotálamo. Além disso, mostramos análise histológica detalhada das células transfectadas até ao nível celular de resolução. Uma comparação de electroporação no útero com outras abordagens para transfecção do cérebro do rato em desenvolvimento in utero pode ser encontrado na secção Discussão.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Sanofi GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>HDG9623</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Ketamin</td>
			<td>Pharma GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Fluka</td>
			<td>44715</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td>&nbsp;</td>
			<td>non-steroidal anti-inflammatory</td>
		</tr>
		<tr>
			<td>Braunoderm</td>
			<td>Braun</td>
			<td>3887138</td>
			<td>povidone-iodine</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline PBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temgesic (buprenorphine)</td>
			<td>Essex Pharma</td>
			<td>&nbsp;</td>
			<td>opioid analgesic</td>
		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Pan-Ophtal</td>
			<td>7136926</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Anaesthetic Device Komesaroff Mark-5</td>
			<td>Medical Developments Australia</td>
			<td>ACN 004 903 682</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Capillary puller P-97</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compresstome</td>
			<td>Precisionary Instr.</td>
			<td>VF-300</td>
			<td>Vibratome-type device</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY21EDIT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrode 1</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY550-10</td>
			<td>Stainless Steel Needle Electrode, 10 mm-Tip, 0. 5 mm diam.</td>
		</tr>
		<tr>
			<td>Electrode 2</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY700P4L</td>
			<td>Cover Round Platinum Plate 4 mm diameter</td>
		</tr>
		<tr>
			<td>Fiberoptic cold light source</td>
			<td>Leica</td>
			<td>KL2500 LCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC120T-15</td>
			<td>1. 2 mm O.D. x 0. 94 mm I.D.</td>
		</tr>
		<tr>
			<td>Glass bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>FST250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Harvard Apparatus</td>
			<td>py872-5272</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Injection device</td>
			<td>World Precision Instruments</td>
			<td>Pneumatic Pico Pump PV820</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Suture Thread Coated Vicryl</td>
			<td>Ethicon</td>
			<td>V4914</td>
			<td>Peritoneal Suture</td>
		</tr>
		<tr>
			<td>Suture Thr. Supramid</td>
			<td>Serag Wiessner</td>
			<td>TO07171L</td>
			<td>Skin Suture</td>
		</tr>
	</tbody>
</table>

genetic manipulation of the mouse developing hypothalamus through in utero electroporation

A modificação genética das regiões específicas do cérebro de mamíferos é o desenvolvimento de uma abordagem experimental, muito poderoso. No entanto, a geração de novos mutantes do mouse é muitas vezes frustrantemente lento. Demonstrou-se que o acesso ao cérebro do rato em desenvolvimento in utero com a sobrevivência pós-operatório é razoável possível. Ainda assim, os resultados com este procedimento foram relatados quase exclusivamente para a parte mais superficial e facilmente acessível do cérebro em desenvolvimento, ou seja, o córtex. O tálamo, uma região mais estreita e mais medial, tem se mostrado mais difícil de atingir. Transfecção em núcleos mais profundo, especialmente aqueles do hipotálamo, é talvez o resultado mais difíceis e, por conseguinte, muito poucas têm sido relatados. Aqui demonstramos um procedimento para atingir todo o neuroepithelium hipotálamo ou parte dela (regiões do hipotálamo) para transfecção por eletroporação. As chaves para a nossa abordagem é mais narcose tempos, injeção na third ventrículo, e tipo e posicionamento dos eletrodos apropriada. Além disso, mostramos resultados de segmentação e posterior análise histológica do núcleo hipotalâmico mais rebaixado, o corpo mamilar.

A maioria dos neurônios do hipotálamo nascem entre E11.5 a E15.2, de acordo com análise de nascimento-dating no rato 26 traduzido para o desenvolvimento um pouco mais curto do rato 27,28. O pico de neurogênese hipotálamo é atingido a E12.5 29-31. Por conseguinte, com a idade de transfecção escolhido para o presente estudo (E12.5), uma grande proporção dos neurónios hipotalâmicos podem ser marcados em qualquer nível rostro-caudal. Análise em E18....

Watch this Scientific Journal Video about Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation at JoVE.com

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Apesar da importância funcional e clínico do hipotálamo,<em&gt; In utero</em&gt; Manipulação genética do seu desenvolvimento tem sido raramente tentada. Mostramos um procedimento detalhado para<em&gt; In utero</em&gt; Eletroporação no hipotálamo do rato e mostram resultados representativos (regional) transfecção hipotálamo total e parcial.

Manipulação genética do rato em desenvolvimento Hipotálamo através<em&gt; In utero</em&gt; Eletroporação

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

genetic-manipulation-mouse-developing-hypothalamus-through-utero

Research

JoVE Journal

Neuroscience

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

15.7K visualizações.  University of Heidelberg. Apesar da importância funcional e clínico do hipotálamo, In utero Manipulação genética do seu desenvolvimento tem sido raramente tentada. Mostramos um procedimento detalhado para In utero Eletroporação no hipotálamo do rato e mostram resultados representativos (regional) transfecção hipotálamo total e parcial.

Vídeo: Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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 ...

Neurociência

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Mouse No Utero Eletroporação: Transfecção Gênica Controlled Spatiotemporal

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualização e manipulação genética de dendritos e espinhas no córtex cerebral e hipocampo do rato usando<em&gt; In utero</em&gt; Eletroporação

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Manipulação genética de Cerebelar Granule Neurônios<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Estudar Neuronal Morfologia e Migração

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Específicos do Striatum Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- e<em&gt; In Utero</em&gt; A electroporação em ratinhos C57BL / 6 mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

In Utero Eletroporação Abordagens para estudar a excitabilidade neuronal subpopulações e Conectividade uma única célula

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

Imaginação confocal de lapso de tempo de neurônios migratórios na cultura de fatias organotípicas do cérebro de mouse embrionário Usando<em&gt; Em Utero</em&gt; Electroporação

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Induzindo a recombinação de Cre-lox no córtex Cerebral de rato através da eletroporação no Utero

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Cirurgia estereotáxica para manipulação genética em células estriadas de cérebro de rato Neonatal

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...