Este trabalho foi financiado por verbas de pesquisa NIH K01AR053210, R01AR061484 e uma bolsa da Fundação Nacional de Psoríase.

1. Informação de fundo A levedura sistema de hibridação é uma poderosa técnica genética usado para descobrir interações proteína-proteína 16, 17. Vários tipos de sistemas híbridos de 2, como LexA sistemas baseados, o Sistema de Recrutamento Sos e bactérias ou células de mamíferos baseadas em dois híbridos, são comerciais disponíveis, este trabalho concentra-se especificamente sobre as modificações dos mais comumente usados ​​GAL4 base sistema de levedura 2-híbrido. Resumidamente, o método é baseado nas propriedades da proteína de levedura que consiste GAL4 de domínios separáveis ​​responsável pela ativação do DNA-binding e transcricional. A proteína isca é expressa como uma fusão para o GAL4 domínio de ligação ao DNA (DNA-BD), enquanto as proteínas presas são expressos como fusões para o domínio de ativação GAL4 (AD). Interação entre isca e proteínas de fusão presa leva à transcrição de ativações GAL4-binding sites que contêm genes repórter que estão integrados no g de fermentoenome. O princípio da Y2H é ilustrado na figura. 1 e do procedimento experimental está resumido na figura. 2. 2. Materiais necessários e soluções <ol><li> Meio de Crescimento YPD (uma mistura de peptona, extrato de levedura e dextrose em proporções ideais para o cultivo de cepas mais Saccharomyces cerevisiae). </li><li> Bases mínima SD (Minimal sintética definida (SD) bases incluem uma base de nitrogênio levedura, sulfato de amônio, e uma fonte de carbono, dextrose. Dropout (DO) suplementos podem ser adicionados à Base SD mínima para fazer uma média, sintético definido sem o especificado nutrientes). </li><li> Leu /-Trp Dropout (DO) complementar (que contém todos os aminoácidos essenciais, exceto para leucina e triptofano) </li><li> -His/-Leu/-Trp/-Ura Dropout (DO) Suplemento (contendo todos os aminoácidos essenciais, exceto para leucina, triptofano, histidina, e uracil) </li><li> Luria caldo (LB) (Triptona 10 g / L, extrato de levedura 5 g / L, NaCl 5 g / L) </li><li> X-Gal (5-bromo-4-cloro-3-indolil-β-D-galactopiranosídeo) solução: preparado como um de 20 mg / ml de solução em N, N-Dimetilformamida </li><li> TE 10X (100 mM Tris-HCl (pH 7,5), 10 mM EDTA, autoclavado) </li><li> Herring sonicado ou DNA do esperma de salmão, cozido (10 mg / ml) </li><li> LiAc 10X (1 acetato de lítio M, autoclavada) </li><li> 50% PEG-3350, solução esterilizada por filtração, </li><li> 3-Amino-1, 2, 4-triazol (3AT) </li><li> Canamicina </li><li> Ampicilina </li><li> Células ELECTROMAX DH5α </li><li> Z buffer: 16,1 g Na2HPO4 7H2O (ou 8,52 g anidro), 5,5 g NaH2PO4 H2O (ou 4,8 ganhydrous), 0,75 g KCl, 0,246 g MgSO4 7H2O (ou 0,12 g anidro), dissolvido em 1 L de água, autoclavada e destilada ajustada para pH 7.0. </li></ol> 3. Geração de isca (pDBLeu-PGRN) Um fragmento de cDNA encoding PGRN falta peptídeo sinal (aa21-588) foi clonado direcionalmente para o I-Não Sal I locais do vetor pDBLeu (a ProQuest sistema de dois híbridos, Invitrogen),mantendo o mesmo quadro de leitura da tradução como o domínio de DNA GAL4 Binding para gerar pDBLeu-PGRN. <ol><li> Amplificação do fragmento de cDNA de PGRN descrito acima por PCR usando primers projetado para conter sítios de restrição (Sal I no 5&#39;end e não eu ao 3&#39;end) para permitir que no quadro-fusion. </li><li> Gel purificar o produto da PCR, digestão com endonucleases Sal I e ​​Not I. Prepare o DB vector pDBLeu restrição por digestão dupla com o endonucleases de restrição mesmo. </li><li> Ligadura do fragmento PGRN restrição na pDBLeu linearizada vetor e transformar em DH5α com a seleção para LB + canamicina a 25 mcg / ml. </li><li> Verificar a correcção da construção de seqüenciamento de DNA. </li></ol> 4. Transformação de pequena escala de isca plasmídeo <ol><li> Inocular 5 ml de YPD com uma colônia de Mav203 (Invitrogen), agitar durante a noite a 30 ° C. </li><li> Diluir a cultura durante a noite em 50 ml de YPD. Crescer um additional 2-4 horas. </li><li> Agregar as células a 3000 rpm por 5 min à temperatura ambiente. Ressuspender o sedimento em 40 ml de água, autoclavada destilada. </li><li> Re-agregar as células. Ressuspender em 2 ml de solução I, incubar a RT 10 min. Solução I: 0,5 ml 10xLiAc, 0,5 ml 10xTE, 4 ml H 2 O </li><li> Dispensar o DNA de tubos: 2-3 mL de pDBLeu-PGRN (0.25μg/μl) e 10 ml de desnaturado DNA do esperma, cortado salmão (10μg/μl). Adicionar 100μl células de levedura, misturar bem. </li><li> Adicionar 700μl de solução II, misture bem. Incubar a 30 ° C por 30 min. Solução II: 0,2 ml 10xLiAc, 0,2 ml 10xTE, 1,6 ml 50% PEG3350. </li><li> Choque térmico a 42 ° C por 15 min. </li><li> Pellet por 2 min. Desprezar o sobrenadante. Ressuspender o sedimento em 200 mL de água, autoclavada destilada. </li><li> Placa de suspensão para SD-Leu placas com diluições em série. Incubar as placas a 30 ° C por 2-3 dias. </li></ol> 5. Validação Bait Antes de executar simª tela de dois híbridos, teste pDBLeu-PGRN para a auto-ativação e determinar os níveis de expressão basal do gene repórter HIS3. Este teste determina se iscas ativam a transcrição e se a auto-ativação pode ser neutralizada por inibidores. 3-Amino-1, 2, 4-triazol (3-AT) é um inibidor competitivo do produto HIS3 gene e pode ser usado para titular o nível mínimo de HIS3 expressão necessária para o crescimento em histidina deficiente mídia. <ol><li> Transformar o pDBLeu-PGRN em cepa de levedura Mav203, placa a transformação para SD-Leu placas e incubar por 48-72 h, a 30 ° C. </li><li> Colônias de patch de cada transformação usando palitos de dente autoclavado para SD-Leu-Seu placas contendo 3-AT em concentrações de 10 mM, 25 mM, 50 mM, 75 mM e 100 mM. 3-AT é um inibidor competitivo da enzima HIS3 e iscas apenas que exibem auto-ativação será capaz de crescer na presença dos 3-AT. </li><li> Cepas isca que crescem em placas containing mM 100 3-AT não são adequados para uso na tela dois híbridos. Para iscas que podem ser usados, use o mais baixo 3-AT concentração que inibe o crescimento celular. Em muitos casos, 25 mM de 3-AT é usado para a seleção. </li></ol> 6. triagem biblioteca de cDNA O pDBLeu-PGRN plasmídeo é introduzido MaV203 usando uma transformação em pequena escala, como descrito acima. Introduzir pPC86-biblioteca (Invitrogen) em MaV203 (pDBLeu-PGRN), o procedimento descrito abaixo tipicamente dá ~ 4 x 10 4 colônias com 0,5 mg de DNA plasmídeo biblioteca. Portanto, 2,5 x 10 6 transformantes de levedura exigirá ~ 30,0 mg pPC86-cDNA DNA plasmídeo biblioteca, 25 transformações, e cinqüenta placas de 10 cm (SD-Trp-Leu-His-Ura 3 AT). <ol><li> Prepare o número apropriado de 10 cm 3 SD-Trp-Leu-His-Ura AT placas (50 para o procedimento abaixo). Também preparar pelo menos quatro de 10 cm SD-Leu-Trp placas para estimar o número de transformantes. </li><li> Transformar a biblioteca emo MaV203 contendo pDBLeu-PGRN de acordo com o procedimento descrito abaixo. <ol><li> Suspender várias colônias isoladas de MaV203 (pDBLeu-PGRN) em ~ 100μl de água, destilada autoclavada e espalhá-los em uma placa de 10 cm SD-Leu. Repita o procedimento para uma placa SD-Leu segundo. Incubar ambas as placas por 18-24 horas a 30 ° C. </li><li> Raspe e completamente suspender as células em 10 ml de água, autoclavada destilada. Adicionar um volume suficiente de suspensão celular a 500 ml de meio YPD líquido num balão de dar uma OD de 600 ~ 0,1. </li><li> Verifique se o OD é de aproximadamente 0,1 após a inoculação. </li><li> Agite a 30 ° C até o OD atinge 600 ~ 0.4. </li><li> Prepare frescos: <ol type="a"><li> 110 ml 1X TE / LiAc através da combinação de 11 ml TE 10X, 11 ml LiAc 10X, e 88 ml de água autoclavada. </li><li> 16 ml PEG / LiAc combinando 1,6 ml TE 10X, 1,6 ml LiAc 10X, e 12,8 ml 50% PEG-3350. </li></ol></li><li> Agregar as células em 3000g por 5 min à temperatura ambiente. </li><li> Descartar tele sobrenadantes e delicadamente ressuspender o sedimento por pipetagem cima e para baixo em 100 ml de água, autoclavada destilada à temperatura ambiente. </li><li> Centrifugar a 3.000 g por 5 min à temperatura ambiente. </li><li> Desprezar o sobrenadante das células centrifugadas e ressuspender o pellet celular em 50 ml 1X solução TE / LiAc. </li><li> Centrifugar a 3.000 g por 5 min à temperatura ambiente. </li><li> Remover o sobrenadante e ressuspender o sedimento em um volume final de 2,5 ml de 1X solução TE / LiAc. </li><li> Realizar 25 transformações. Combine 2,5 células ml, 125 mL (10μg/μl) desnaturados, DNA de esperma de salmão cortado, e 30 mg biblioteca de cDNA. Misture suavemente pipetando cima e para baixo. Adicionar 15 ml de solução de PEG / LiAc e misture delicadamente. Alíquota em 25 autoclavado tubos de 1,5 ml de microcentrífuga de 700 mL cada. </li><li> Incubar por 30 min em banho-maria a 30 ° C. </li><li> Choque térmico durante 15 min em banho-maria a 42 ° C. </li><li> Centrifugar a 6.000 g por 1 min em temperatura ambiente. Remova cuidadosamente o supersobrenadante. Delicadamente ressuspender cada pellet em 400 mL de água, destilada autoclavada por pipetagem cima e para baixo. </li></ol></li><li> Placa de 200 mL da mistura de transformação em cada transformação única de 10 cm de SD-Trp-Leu-His-Ura 3 AT (3AT concentração: 25 mM) placas usando uma barra de spread, de modo 25 autoclavado 1,5 ml tubos de microcentrífuga pode fazer 50 placas (SD-Trp-Leu-His-Ura 3 AT). </li><li> Incubar as placas para os dias 10/05 a 30 ° C. </li><li> Para estimar a eficiência de transformação da reação, diluições em série de uma reação (1:40; 1:400 e 1:4000) são semeadas em SD-Leu-Trp prato. Após incubação por 3 dias a 30 ° C, o número de colônias é contado eo número total de transformantes calculado. </li></ol> 7. X-gal ensaio <ol><li> Transferência de colônias para placa de YPD; incubar a 30 ° C durante a noite. </li><li> Lugar arredondado membrana de nitrocelulose na placa de YPD, certifique-se que não há bolhas entre a membrana ea placa de YPD. Após 1-2 min, make se todas as colônias são transferidos para membranas (papel de nitrocelulose). </li><li> Coloque o lado colônia de membrana em barco feito com papel de alumínio. </li><li> Colocar a membrana em nitrogênio líquido, esperar 20 segundo, e submergir em nitrogênio líquido por alguns minutos. </li><li> Retire membrana para descongelar. </li><li> Enquanto isso, misture o reagente seguinte: 1,5 ml de buffer Z 20μl X-gal (20 mg / ml) </li><li> Queda Z buffer / X-gal, numa placa de Petri, coloque papel Whatman em cima disso. </li><li> Coloque cuidadosamente o papel de nitrocelulose em cima de papel Whatman. </li><li> Incubar a 37 ° até colônias azuis aparecem. </li></ol> 8. Ensaio retransformação Proteínas de fusão Prey (AD-Y) isolados de triagem biblioteca deve manter a interação com a proteína de fusão isca (DB-X) para induzir os genes relatório, e os clones retransformação de rapina e isca construir em levedura pode ainda eliminar falsos positivos e facilitar adicionaranálise itional. <ol><li> Isolar DNA plasmidial de cepas de leveduras potencialmente contendo proteínas que interagem. </li><li> Transformar DNA em células de E. coli DH5α, a chapa do transformações no LB 100 mg / ml ampicilina placas para seletivamente isolar a biblioteca pPC86-cDNA, e incubar durante a 37 ° C. </li><li> Escolha diversas colônias da placa para colocar em LB 100 mcg / ml caldo de ampicilina. </li><li> Prepare miniprep DNA e examinar por análise de restrição de casal com Sal I e ​​Not I. </li><li> Co-transform a presa plasmídeo e isca plasmídeo em MaV203, a chapa do misturas de transformação na SC-Leu-Trp placas e incubar durante 2-3 dias a 30 ° C. </li><li> Escolher três diferentes colônias da placa SC-Leu-Trp, execute X-gal de Ensaio. </li></ol> 9. Seqüenciamento e análise bioinformática Seqüenciar o DNA plasmídeo que foi isolado provavelmente verdade clones positivos, compare essas seqüências aos do GenBank utilizando o BLAST programa, e identificar os clones de dois híbridos, que correspondem a genes conhecidos. Dados de sequenciamento mostrou que dois dos 12 positivos obtidos foram acima da superfície celular TNFR2 (TNFRSF1B/CD120b; Adesão # NM_130426). Além disso, a interação entre PGRN e TNFR foi verificada usando vários ensaios de interação proteína-proteína, incluindo in vitro em fase sólida ensaio de ligação, Co-imunoprecipitação, plasmon de superfície análise de ressonância, e ensaio de citometria de fluxo 14. 10. Resultados representante O fluxograma da triagem é descrito na fig. 3. Normalmente 50-100 candidatos clone positivo será obtido nesta etapa. Inicialmente isolados 54 candidatos clone positivo entre 2,5 milhão transformantes selecionados com a isca PGRN. Candidatos clone positivos foram verificados, em seguida, através da realização de X-gal ensaio. Normalmente, cerca de 50% de falsos positivos clones são removidos através de X-gal ensaio. Obtivemos 23 clones positivos candid<html> ates nesta etapa para a isca PGRN (Fig. 4). Retransformação dos clones presa e construção de isca para a levedura ainda eliminar falsos positivos e clones que ainda ativar os genes repórter provavelmente representam os verdadeiros positivos. Normalmente, cerca de 50% candidatos clone positivo será removido. Finalmente isolado 12 clones positivos que interagem com PGRN em levedura. <img alt="Figura 1" src="/files/ftp_upload/3562/3562fig1.jpg" /> Figura 1. Princípio da levedura sistema de dois híbridos <img alt="Figura 2" src="/files/ftp_upload/3562/3562fig2.jpg" /> Figura 2. Pipeline de identificação de parceiros de ligação às proteínas usando o sistema de levedura híbrida de dois <img alt="Figura 3" src="/files/ftp_upload/3562/3562fig3.jpg" /> Figura 3. Fluxograma de levedura triagem dois híbridos biblioteca.rge.jpg &quot;target =&quot; _blank &quot;&gt; Clique aqui para ver uma versão em tamanho normal da imagem. <img alt="Figura 4" src="/files/ftp_upload/3562/3562fig4.jpg" /> Figura 4. Ensaio de beta-galactosidase de candidatos clone positivo. A clones, positivos obtidos a partir do ecrã biblioteca foram transferidas para placa de YPD e incubadas a 30 ° C durante a noite; B, Todas as colônias na chapa YPD foram transferidas para membranas de nitrocelulose e beta-galactosidase ensaio realizado.

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Triagem levedura híbrida de dois provou ser uma ferramenta eficaz na identificação de interação proteína 16, 17. Em comparação com outras abordagens para a identificação de proteínas de ligação de parceiros, como a bioquímica co-purificação de proteínas e chips, levedura híbrida de dois sistemas é uma abordagem sensível genética que pode ser usado para screening um grande número de seqüências de codificação em um experimento relativamente simples, em Além disso, ele detecta a intera...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> ProQuest sistema de dois híbridos </td><td> Invitrogen </td><td> 10835 </td></tr><tr><td> YPD Meio de Crescimento </td><td> Clontech </td><td> 630409 </td></tr><tr><td> YPD Agar Médio </td><td> Clontech </td><td> 630410 </td></tr><tr><td> Base de Dados de agar mínimo SD </td><td> Clontech </td><td> 630412 </td></tr><tr><td> -Leu DO Suplemento </td><td> Clontech </td><td> 630414 </td></tr><tr><td> -Leu/-Trp DO Suplemento </td><td> Clontech </td><td> 630417 </td></tr><tr><td> -His/-Leu/-Trp/-Ura DO Suplemento </td><td> Clontech </td><td> 630425 </td></tr><tr><td> 3-Amino-1 ,2,4-triazol (3AT) </td><td> Sigma-Aldrich </td><td> A8056 </td></tr><tr><td> Luria caldo (LB) </td><td> Sigma-Aldrich </td><td> L3022 </td></tr><tr><td> X-Gal </td><td> Invitrogen </td><td> 15520-034 </td></tr><tr><td> DNA de esperma de salmão sonicado </td><td> Stratagene </td><td> 201190 </td></tr><tr><td> Ampicilina </td><td> AMRESCO </td><td> 0339 </td></tr><tr><td> Sulfato de canamicina </td><td> Invitrogen </td><td> 11815-024 </td></tr><tr><td> Eficiência subclonagem ™ DH5α ™ células competentes </td><td> Invitrogen </td><td> 18265-017 </td></tr><tr><td> Trizma ® de base </td><td> Sigma-Aldrich </td><td> T6066 </td></tr><tr><td> Acetato de lítio </td><td> Sigma-Aldrich </td><td> L4158 </td></tr><tr><td> Ácido etilenodiaminotetracético </td><td> Sigma-Aldrich </td><td> ED </td></tr><tr><td> Membrana de nitrocelulose </td><td> Bio-Rad </td><td> 162-0115 </td></tr><tr><td> 10 cm de placa de Petri </td><td> ITI Scientific </td><td> CT-903 </td></tr><tr><td> Incubadora (30 ° C) </td><td> ATR (Ecotron) </td><td></td></tr></table>

modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin

Progranulina (PGRN), também conhecido como precursor granulin epithelin (GEP), é um fator de crescimento 593-amino-ácido autócrina. PGRN é conhecida por desempenhar um papel crítico em uma variedade de processos fisiológicos e de doença, incluindo a embriogênese precoce, uma cicatrização de feridas, inflamação 2, 3 e 4 de defesa do hospedeiro. PGRN também funciona como um fator neurotrófico 5, e as mutações no gene PGRN resultando em perda parcial da demência frontotemporal causa proteína PGRN 6, 7. Nossos estudos recentes têm levado ao isolamento de PGRN como um importante regulador da cartilagem desenvolvimento e degradação 8-11. Embora PGRN, descoberto há quase duas décadas, desempenha um papel crucial em várias condições fisiológicas e patológicas, os esforços para explorar as ações de PGRN e entender os mecanismos envolvidos foram significativamente prejudicada pela nossa incapacidade de identificar a sua ligação ao receptor (s). Para resolver este problema, desenvolvemos um modicados levedura híbrida de dois (MY2H) abordagem baseada no mais comumente usados ​​GAL4 sistema 2-híbrido baseado. Comparado com o convencional levedura tela dois híbridos, MY2H reduz drasticamente o processo de tela e reduz o número de falsos positivos clones. Além disso, esta abordagem é reproduzível e confiável, e temos empregada com sucesso este sistema para isolar as proteínas de ligação de iscas diversas, inclusive de canal iônico, 12 de proteínas da matriz extracelular 10, 13, e fator de crescimento 14. Neste artigo, descrevemos este procedimento MY2H experimental em detalhe usando PGRN como um exemplo que levou à identificação de TNFR2 conhecido como o primeiro receptor PGRN associada 14, 15.

Watch this Scientific Journal Video about Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin at JoVE.com

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Nós modificamos o fermento convencional de dois híbridos de rastreamento, uma ferramenta eficaz na identificação genética de interação de proteínas. Esta modificação marcadamente encurta o processo, reduz a carga de trabalho, eo mais importante, reduz o número de falsos positivos. Além disso, esta abordagem é reproduzível e confiável.

Modificado Sistema Yeast-Two-Hybrid para identificar proteínas Interagindo com o Fator de Crescimento progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

modified-yeast-two-hybrid-system-to-identify-proteins-interacting

Research

JoVE Journal

Biology

28.6K visualizações.  NYU Hospital for Joint Diseases. Nós modificamos o fermento convencional de dois híbridos de rastreamento, uma ferramenta eficaz na identificação genética de interação de proteínas. Esta modificação marcadamente encurta o processo, reduz a carga de trabalho, eo mais importante, reduz o número de falsos positivos. Além disso, esta abordagem é reproduzível e confiável.

Vídeo: Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads.

Fator-Revestido crescimento Placement Bead em explantes Encéfalo anterior Dorsal

Biologia

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

Fator de Crescimento colocando-Revestido Beads em embriões de galinha Early Stage

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

Preparando-se Fator de Crescimento de Células T esplenócitos Rat

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Proteômica para identificar proteínas Interagindo com P2X2 Canais Ligand-Gated Cation

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-Ubiquitina Yeast Membrana Baseado Two-Hybrid (MITO) do sistema: uma ferramenta poderosa para identificação de interações proteína-proteína

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - um método para identificar em toda a Transcriptoma Sítios de Ligação de Proteínas RNA Binding

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi de triagem para identificar fenótipos pós-embrionário em C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

Reverter levedura Sistema de dois híbridos para identificar mamíferos Receptor Nuclear Resíduos que interagem com ligantes e / ou antagonistas

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Captura de Espectrometria de Massa Compound - uma ferramenta poderosa para identificar novos c-di-GMP efetoras Proteínas

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&amp;#945; Promoter Using Gibson Assembly

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

Geração de plasmídeo vectores que expressam proteínas marcadas-bandeira nos termos do regulamento de Alongamento Human Fator-1α Promotor Usando Assembleia Gibson

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...