Trabalho no laboratório é apoiado pela NSF (MCB0919933) e NIH (GM088790).

1. Tronco ensaio de regeneração do fragmento <ol><li> Parar de alimentar uma coorte de worms por pelo menos cinco dias antes do ensaio regenerativa para garantir os animais são livres de resíduos e alimentos ingeridos (~ 30 worms intactos, cada minhoca ~ 8-10 mm de comprimento pós-inanição). </li><li> No dia do ensaio, lavar um prato pré-congelado de gelo nivelado com água e cobrir a superfície plana gelada com filme plástico. Utilizando uma pinça, coloque um filtro de papel sobre o plástico. </li><li> Umedecer o papel de filtro com algumas gotas de água de nascente. Transferência de planárias em filtro (&lt;20 worms por filtro), utilizando uma pipeta. Worms pode ser reposicionada no filtro, se necessário, por repipetting com água mais primavera. Retire o excesso de fluido. </li><li> Usando um bisturi, amputar a cabeça por um único corte feito aproximadamente a meio caminho entre o ápice anterior do animal e da extremidade anterior da faringe (Figura 1A). </li><li> Usando um bisturi, amputar a região da cauda por um único corte feito aproximadamente a meio caminho entre o fim da cauda do animal e extremidade posterior da faringe (Figura 1A). Retire o excesso de muco no bisturi usando uma toalha de papel umedecido com álcool 70% após o corte. Limpe o excesso de etanol bisturi. </li><li> Transferência do filtro através de pinças em uma placa de Petri (100 x 25mm profundidade) contendo água de nascente. Espere ~ 3 minutos (para o fechamento da ferida, observável por um beliscar e escurecimento da ferida). </li><li> Enxágüe tronco a partir de fragmentos de papel de filtro para placa de Petri contendo meio desejado para o ensaio de regeneração e transferência para termostatizado incubadora (24 ° C). </li><li> Pontuação fenótipo regenerativa após ~ 1 semana, quando as estruturas regeneradas são identificáveis ​​(Figura 1A). </li></ol> 2. Manipulação farmacológica da regeneração: bipolaridade induzida por praziquantel <ol><li> Prepare estoque praziquantel (PZQ) por droga solubilização em DMSO (62.4mg PZQ em 1mL de ações 200mM). Use no mesmo dia. </li><li> Faça 50mL de drogas contendo solução (90 mM PZQ) em sais Montjuch buffered. Vortex de ~ 1 minuto para garantir a dispersão. </li><li> Realizar ensaio de tronco regenerativas fragmento como detalhado acima [protocolo n º 1], expondo uma coorte de fragmentos tronco para PZQ na etapa final [1,7]. </li><li> Após 24-48 horas, a troca PZQ contendo mídia para água mineral, lavar várias worms (&gt; 3) vezes. </li><li> Retorno worms para incubadora. Bipolaridade pontuação (dois-cabeças, Figura 1B) uma semana após o corte. </li></ol> 3. Manipulação genética de regeneração: in vivo protocolo de alimentação RNAi <ol><li> Antes do ensaio, prepare homogeneizado de fígado de galinha. Descartar graxos, amarelo seções coloridas de estoque de alimentos, e purê de fígado remanescente. Filtro de puré através da malha do filtro e suspensão alíquota para armazenamento (1,5 ml tubos, -20 ° C). </li><li> Antes do ensaio, prepare bovina glóbulos vermelhos (hemácias) por alíquotas de fornecimento em amostras de 1 mL, armazenadas a -20 ° C. </li><li> Selecione worms para RNAi. Três coortes são usados: o gene de interesse, controle positivo (gene com fenótipo RNAi conhecidos; Figura 1C, D &amp; E) e controle negativo (gene sem fenótipo RNAi, também útil para avaliação dos níveis de knockdown em relação ao grupo experimental). Para cada coorte, use ~ 250 worms (~ 8-10 mm de comprimento após passar fome durante pelo menos 5 dias). Loja em água de nascente na banheira de plástico. </li><li> Para cada RNAi estoque descongelar coorte, de E. coli expressando dsRNA gene targeting de interesse [8], juntamente com um tubo de homogenato de fígado de frango [3.1] e um tubo de hemácias [3.2]. </li><li> Pellet de bactérias (13.000 g, 1 minuto). Remover o sobrenadante e ressuspender sedimento em 2xYT media (~ 700μl). Recentrifuge, elimine o sobrenadante, coloque pellet no gelo. </li><li> Criar um grande furo P200 ponteira cortando o final da ponta. Completamente ressuspender o sedimento de bactérias em uma mistura de fígado de galinha homogenato (150 mL) e hemácias (50 mL) para criar RNAi mix de alimentação. Remover bolhas de ar por centrifugação. </li><li> Para a alimentação, retire a maior parte da água da banheira de plástico contendo worms (leave ~ profundidade de 1 polegada). Tub redemoinho de concentrar worms no centro deste recipiente e misturar a alimentação pipeta RNAi em um círculo corralling worms. Use uma pipeta com cuidado coaxial fugitivos de volta para o mix de alimentação RNAi sem perturbar outros clientes! </li><li> Após a alimentação (~ 1 hora), identificar planárias que alimentou bem. Esses vermes mostram uma coloração vermelho profundo de hemácias ingerido. Descartar outros. </li><li> Recolocá alimentação solução com água fresca, minimizando distúrbios para evitar egestion de alimentos. Para fazer isso, gentilmente localizar os worms para um lado do tubo com uma pipeta de transferência, derramar água turva, e recolocá com água fresca derramadas na parede oposta da banheira. Planárias Resubmerge mais rapidamente a exposição prolongada ao ar também resulta em egestion alimentos. </li><li> Vagamente sial tampa do recipiente e retornar à incubadora (24 ° C) </li><li> Repita RNAi protocolo de alimentação durante vários ciclos (~ 2-3 dias de intervalo), intercalados com ciclos regenerativos [protocolo # 1] para triagem de fenótipos RNAi. Um protocolo padrão de alimentação e de regeneração eficaz para muitos genes é mostrado na Figura 2, que leva ~ 1 mês de duração total. </li></ol> Modificar este esquema para genes diferentes, como protocolo ideal dependerá de fatores como a estabilidade do RNAm, a localização do tecido, perdurance proteína, ou o desenvolvimento de um fenótipo que impede os ciclos de alimentação múltiplas após a regeneração. Avaliar os níveis de knockdown simplesmente triagem para penetrância do fenótipo (se aparente), ou por abordagens qPCR comparar os níveis de mRNA alvo de grupo de controlo negativo. 4. Resultados representativos: O tronco ensaio regeneração fragmento é tão forte que todos os vermes deve regenerar normais ântero-posterior (&quot;cabeça à cauda) polaridade. Isto pode ser marcado assim que cinco dias após o corte, facilitada pelo surgimento da eyespots anterior (asterixed, Figura 1A). No entanto, a restauração completa das estruturas morfológicas perdido ocorre após uma semana. Manipulação farmacológica da regeneração fragmento de tronco por PZQ para produzir duas cabeças worms (Figura 1B) também é robusta (EC 50 = 87 (+ -) 11% fragmentos bipolar, 70μM PZQ por 48 horas 20). Bipolaridade ocorre ao longo de um único ciclo regenerativo. Menor eficácia nestes ensaios provavelmente relacionado a problemas com a solubilidade de drogas e / ou armazenamento, ou o uso de uma espécie diferente, onde platelminto PZQ é ineficaz. De drogas com menor penetrância, um índice anteriorização é muitas vezes usado para marcar fenótipos intermediários para além de 22 bipolaridade completa. Fenótipos resultantes de RNAi de construções de controle positivos são mostrados na Figura 1: RNAi de Dugesia japonica seis-1 (D j e seis-1) resulta em um fenótipo eyeless 23 (Figura 1C), RNAi de Dugesia PC2 japonica (Dj-PC2) resulta em uma perda da resposta aversão a luz 24 (Figura 1D) e RNAi de Dugesia japonica βcatenin-1 (Dj-βcatenin-1) resulta em um fenótipo de duas cabeças 16-19, 21 a partir de fragmentos do tronco (Figura 1E). Estes fenótipos pode ser conseguido usando os seguintes protocolos de RNAi simples: Dj e seis-1 (FFxFx), Dj-PC2 (FFFx), Dj-βcatenin-1 (FFxFx), onde F = ciclo de alimentação e x = ciclo regenerativo, respectivamente. <img src="/files/ftp_upload/3058/3058fig1.jpg" alt="Figura 1"/> Figura 1:... Trunk ensaio de regeneração fragmento de A Esquerda, Imagem do planária (topo) e esquema (meio) para mostrar a posição do fragmento retirado do tronco direito, timecourse de regeneração fragmento de tronco mostrando a aparência de um fragmento de regeneração, por vezes indicado (dias) B imagem de duas cabeças planária produzida pela exposição PZQ. verme C &#39;Eyeless &quot;produzido pelo Dj e seis-1 RNAi. D Perda de resposta de aversão a luz no imóvel Dj-PC2 RNAi worms (manchada de vermelho), em comparação com o controle sem-fim móvel (manchada verde). E Bipolar planária produzido por Dj-βcatenin-1 RNAi. No BE, o final original anterior dos vermes RNAi é orientado para a esquerda. <img src="/files/ftp_upload/3058/3058fig2.jpg" alt="Figura 2"/> Figura 2: Seqüência de ciclos de alimentação e corte de protocolo de RNAi típico composto por múltiplas refeições (F), e os ciclos de regeneração (x) que é eficaz para knockdown de muitos genes em D. japonica. Modificação do presente protocolo para genes individuais pode ser necessária para produzir os melhores resultados, por exemplo usando um arquivo. Menores (entre parênteses) ou um número maior de alimentação e ciclos de regeneração

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Não há conflitos de interesse declarados.

Os protocolos descritos aqui detalhadamente os ensaios para o estudo e manipulação de regeneração da planária Dugesia japonica. Eles são simples e não requerem equipamento especializado de modo que possam ser facilmente realizado no laboratório ou sala de aula. Ensaios podem ser realizados individualmente ou combinados (químicos para rastreamento genético de eficácias de drogas in vivo) e pode ser realizada ao nível de genes candidatos, ou são adaptáveis ​​a throughput screening imparcial, acima de 8. Se para o estudo da biologia de planárias intrigante em seu próprio direito, ou para a avaliação in vivo em função de homólogos mamíferos em um modelo alternativo para estudar a regeneração do tecido, estas abordagens devem catalisar o interesse de uma gama diversificada de pesquisadores.

<table border="1"><tbody><tr><th> Reagente </th><th> Vendedor </th><th> Número Catálogo </th><th> Comentários </th></tr><tr><td> Água de nascente </td><td> Kandiyohi. Premium Waters Inc. Minneapolis, MN </td><td> n / a </td><td> Outras formas de água de nascente funcionar bem também. Primeiro julgamento em ensaios de viabilidade. </td></tr><tr><td> 1 x buffered Montjuich sais: NaCl (1,6 mm), CaCl2 (1mM), MgSO4 (1mM), MgCl2 (0,1 mM), KCl (0,1 mm), NaHCO3 (1,2 mm), HEPES (1,5 mM). pH 7,4 a 24 ° C. </td><td> Múltiplos fornecedores </td><td> n / a </td><td> Água artificial para os tratamentos com drogas durante os ensaios de regeneração para garantir buffer pH. Solução 08/05 Holtfreter é uma alternativa. </td></tr><tr><td> 2xYT Caldo </td><td> Fisher Scientific </td><td> BP2467-500 </td><td> Media = 31 g / L. Autoclavado. </td></tr><tr><td> Petri Dish (100x25mm) </td><td> Fisher Scientific </td><td> 08-757-11 </td><td> Worms habitação durante os ciclos de regeneração </td></tr><tr><td> Praça Dish (100x100x15mm) </td><td> Fisher Scientific </td><td> 08-757-11A </td><td> Encha com água, para congelar bandeja de gelo usado como superfície de corte verme </td></tr><tr><td> Tubo de plástico: Twist Ziploc &#39;n Loc (16 onças). </td><td> Vários retalhistas </td><td> n / a </td><td> Conveniente recipientes de água apertado para coortes RNAi </td></tr><tr><td> Fígado de frango </td><td> Mantimento comercial </td><td> n / a </td><td> Viés para suprimentos orgânicos, devido à evasão de antibióticos. </td></tr><tr><td> Lado Blender </td><td> Qualquer fornecedor de cozinha </td><td> n / a </td><td> Use para fazer purê de fígado de galinha </td></tr><tr><td> Fio de 1 milímetro de malha do filtro </td><td> Qualquer fornecedor de cozinha </td><td> n / a </td><td> Use para esticar purê de fígado de galinha </td></tr><tr><td> Bovina glóbulos vermelhos </td><td> Lampire Biological Laboratories </td><td> 7240807 </td><td> 100% lavado e pooled suspensão RBC </td></tr><tr><td> Circular papéis de filtro </td><td> Whatman # 3 </td><td> 1003 055 </td><td></td></tr><tr><td> Transferência Pipetar </td><td> Fisher Scientific </td><td> 13-711-41 </td><td></td></tr><tr><td> Estéreis, lâminas cirúrgicas </td><td> Múltiplos fornecedores </td><td> n / a </td><td></td></tr><tr><td> ± Praziquantel </td><td> Sigma Aldrich </td><td> P4668 </td><td> Alíquotas em pó armazenar no escuro a 4 ° C. Desidratar. </td></tr><tr><td> Dj e seis-1 </td><td></td><td> GenBank AJ557022.1 </td><td> RNAi para controle positivo &quot;olho-less&quot; fenótipo 23 </td></tr><tr><td> Dj-βcatenin-1 </td><td></td><td> GenBank AB181913.1 </td><td> RNAi para controle positivo de duas cabeças fenótipo 17 </td></tr><tr><td> Dj-PC2 </td><td></td><td></td><td> RNAi controle positivo para a perda do fenótipo aversão a luz 24 </td></tr></tbody></table>

pharmacological and functional genetic assays to manipulate regeneration of the planarian dugesia japonica

Platelmintos de vida livre planária tem uma longa história de uso experimental, devido às suas notáveis ​​capacidades regenerativas 1. Pequenos fragmentos retirados de estes animais reformar o plano de corpo original após a regeneração das estruturas do corpo faltando. Por exemplo, se um fragmento &quot;tronco&quot; é cortado a partir de um verme intacta, &#39;cabeça&#39; de um novo vai gerar anteriormente e um &#39;rabo&#39; vai gerar posteriormente restaurar o original polaridade &quot;head-to-tail&quot; das estruturas do corpo antes da amputação (Figura 1A). Regeneração é impulsionado por células-tronco em planárias, conhecido como &quot;neoblasts &#39;que se diferenciam em ~ 30 diferentes tipos de células durante a homeostase corporal normal e regeneração de tecidos aplicadas. Este processo regenerativo é robusto e fácil de demonstrar. Devido à dedicação de vários laboratórios pioneiros, muitas ferramentas e métodos genéticos funcionais já foram otimizados para o sistema modelo. Conseqüentemente, os progressos recentes considerável tem sido feito na compreensão e manipulação dos eventos moleculares subjacentes à plasticidade do desenvolvimento planária 2-9. O sistema modelo planária serão de interesse para uma ampla gama de cientistas. Para os neurocientistas, o modelo oferece a oportunidade de estudar a regeneração de todo um sistema nervoso, ao invés de simplesmente a regeneração / reparação de processo único de células nervosas que normalmente são foco de estudo em muitos modelos estabelecidos. Planárias expressar uma infinidade de neurotransmissores 10, representam um importante sistema para o estudo da evolução do sistema nervoso central 11, 12 e têm potencial de triagem comportamental 13, 14. Regenerativa resultados são passíveis de manipulação por apparoaches farmacológicos e genéticos. Por exemplo, as drogas podem ser rastreados para efeitos sobre a regeneração simplesmente colocando fragmentos do corpo em soluções contendo droga em diferentes momentos após a amputação. O papel de genes individuais pode ser estudada usando métodos knockdown (in vivo RNAi), que pode ser conseguido através de ciclos de microinjeção ou alimentando-bacterially expressa constrói dsRNA 8, 9, 15. Ambas as abordagens podem produzir fenótipos visualmente impressionante em alta penetrância, por exemplo, regeneração de animais bipolar 16-21. Para facilitar a adoção deste modelo e implementação de tais métodos, nós apresentamos neste artigo vídeo protocolos para ensaios farmacológicos e genéticos (RNAi in vivo pela alimentação), utilizando o planária Dugesia japonica.

Watch this Scientific Journal Video about Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica at JoVE.com

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Um modelo atraente para estudar a diferenciação das células-tronco dentro de um animal vivo é o platelminto planária. Regeneração é estudado por experimentos amputação simples que são facilmente realizados em um laboratório básico e são passíveis de (farmacológicos e genéticos<em&gt; In vivo</em&gt; RNAi) manipulação, conforme detalhado por protocolos neste artigo.

Farmacológica e funcional Ensaios genética para manipular Regeneração da planária Dugesia japonica</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

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Biology

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

36.8K visualizações.  University of Minnesota Medical School. Um modelo atraente para estudar a diferenciação das células-tronco dentro de um animal vivo é o platelminto planária. Regeneração é estudado por experimentos amputação simples que são facilmente realizados em um laboratório básico e são passíveis de (farmacológicos e genéticos In vivo RNAi) manipulação, conforme detalhado por protocolos neste artigo.

Vídeo: Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Claro / escuro Teste de transição para Ratos

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Biologia

Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies

Neurogênese cortical: Transição do Avanços no Laboratório de terapias baseadas em células

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Ensaio para Indução Neural no embrião de galinha

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Labirinto elevado para Ratos

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

Dissecção do giro denteado do hipocampo de rato adulto

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

A abordagem inversa Genética para Teste de Redundância funcional durante a embriogênese

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

Um alelo-específico do ensaio Expressão Gênica para testar as bases funcionais das Associações de Genética

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Imobilização planária, irradiação parcial e Transplante de Tecidos

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Mapeamento bacterianas redes funcionais e vias<em&gt; Escherichia Coli</em&gt; Usando sintéticos matrizes genéticas

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

Os pequenos extratos nucleares para ensaios funcionais da expressão gênica Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Farmacológica e funcional Ensaios genética para manipular Regeneração da planária Dugesia japonica

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