Somos gratos à Universidade Estadual de Ohio Comprehensive Cancer Center para a concessão de financiamento para este trabalho.

1. Linha de célula com um substrato Recombinação Integrado <ol><li> As células HeLa foram estavelmente transformada com PDR-GFP (dom 7 plasmídeo de M. Jasin, Memorial Sloan-Kettering Cancer Center) e mantidos em puromicina para a seleção de transformantes. </li><li> As células são passado em DMEM / SFB, contendo 10% de soro fetal bovino, 2 mM Glutamax, piruvato de sódio 1 mM, a penicilina / estreptomicina (100 U / mL/0.1 mg / mL), e 1,5 puromicina ug / mL. </li></ol> O substrato de recombinação na pDR GFP-plasmídeo, e em transformantes estáveis, contém dois alelos inativos da GFP (Figura 1). Um alelo está inativo devido à inclusão em sua seqüência de codificação da restrição bp 18 sítio de reconhecimento da enzima para a I-SCEI. Expressão de I-SCEI nestas células cria uma quebra de DNA de fita dupla (DSB). Há um segundo alelo de GFP inativos nesta DNA, mas a parte de sua seqüência correlacionados com o site I-SCEI sobre o alelo primeira é do tipo selvagem. Este alelo GFP segundo pode funcionar como um doador de seqüência para homólogos de reparo de recombinação do DSB e resultaria em um alelo GFP ativo, que é facilmente detectada por citometria de fluxo. Alternativamente, o DSB pode ser reparado por nonhomologous final de adesão, que iria destruir o sítio de restrição I-SCEI e resultaria em células GFP-negativos. O nível de recombinação homóloga em uma célula é determinado pela contagem da porcentagem de células positivas para GFP-. Esta linhagem celular, pHeLa-DR13-9, foi selecionado entre todos os clones para zero GFP sinal de fundo e uma percentagem relativamente elevada de células positivas para GFP-transfecção em cima com o I-SCEI expressar pCBASce, plasmídeo. (O pCBASce e seu controle de vetores, pCAGGS, foram ambos fornecidos pela M. Jasin do Centro de Câncer Memorial Sloan-Kettering.) A linha HeLa-DR13-9 celular está disponível a partir dos autores, mediante solicitação. 2. Transfecção de Destroem BRCA1 endógena e Add Voltar BRCA1 mutante. <ol><li> O dia antes da transfecção primeiro (geralmente uma terça-feira): Comece com um prato de 10 cm de HeLa-DR13-9 células, pelo menos, 90% confluentes. </li><li> Trypsinize em 1 mL e parar a reacção adicionando 9 mL DMEM / SFB, misture bem por pipetagem. </li><li> Adicionar 40 mL de células tripsinizados por poço de uma placa de 24 poços em 0,5 mL de DME / FBS. </li><li> Dia 1 transfections (quarta-feira): as células devem ser confluentes ~ 40-50%, se muito menos começar de novo. Transfecção através da mistura de: 5 siRNA pmol + 0,3 ug-BRCA1 mutante expressar DNA plasmídeo em 12,5 mL Opti-MEM; e 0,5 mL Lipofectamine 2000 em 12,5 mL Opti-MEM. Prosseguir com a transfecção de acordo com o protocolo Lipofectamine (Invitrogen). </li><li> Substituir a mídia de 4-6 horas mais tarde. O siRNA que usamos para esgotar BRCA1 é baseada na seqüência GCUCCUCUCACUCUUCAGU especificando BRCA1 3&#39;-UTR seqüências de 80.780 para 80.798 (número de adesão AY273801). </li><li> Dia 2: Transferência de células por trypsinizing da placa de 24 poços a uma placa de 6 bem. </li><li> Dia 3 transfections: 25 siRNA pmol + 0,75 ug-BRCA1 mutante expressar DNA plasmídeo + 0,75 ug pCBASce (ou pCAGGS controle de vetores) em 62,5 mL Opti-MEM e 2,5 mL Lipofectamine 2000 em 62,5 mL Opti-MEM. Substituir a mídia de 4-6 horas mais tarde. </li></ol> 3. Análise de Transfectants. <ol><li> No dia 6, trypsinize células de cada poço e parar em 1 mL DMEM / SFB. Analisando as células de expressão GFP pode ser feito nos dias 5 ou 7, mas nós achamos que o dia 6 funciona melhor. </li><li> Analisar 10.000 células por poço por citometria de fluxo. Nós usamos um instrumento Becton Dickinson FACSCalibur na Universidade Estadual de Ohio Comprehensive Cancer Center Citometria Analytical recurso compartilhado. Único, as células vivas são fechados utilizando os parâmetros de dispersão para a frente e lateral. GFP células positivas são detectadas usando o GFP detector de fluorescência (FL1), e os resultados são apresentados como um histograma (Figura 2, abaixo). </li><li> Esquerda sobre as células podem ser replated para a fotografia de microscopia de fluorescência, se desejar. </li></ol> 4. Resultados representativos: BRCA1 regula o caminho para a reparação de DNA de fita dupla breaks por recombinação homóloga 6,11. As células que sofreram reparo por recombinação homóloga são facilmente detectados por microscopia de fluorescência (Figura 2, em cima). Esgotamento dos resultados BRCA1 em uma diminuição da GFP-positivos detectados células (Figura 2, à direita). A saída do FACSCalibur é um histograma com intensidade GFP por célula no eixo X e do número de células no eixo Y (Figura 2, abaixo). Em um conjunto típico de resultados a partir de um único experimento, 15,5% das células foram GFP-positivo após I-SCEI expressão e de esgotamento controle RNAi (Figura 3). Em comparação, o esgotamento dos genes BRCA1 e vector adicionar novamente reduzida GFP-conversão para 0,7%. Add-back de tipo selvagem BRCA1 ou de BRCA1 (I21V) totalmente restaurado recombinação homóloga, detectada através da obtenção de 15-16% de células positivas para GFP. Add-back do BRCA1 (M18T) mutantes (Figura 3, faixa 5) teve efeito semelhante ao add-parte de trás do controle de vetores. Uma vez que esta variante expressa em níveis semelhantes como a proteína endógena 9, que assim interpretar o BRCA1 variante (M18T) não é funcional na recombinação homóloga. Nós normalmente têm 10-20% das células se converter ao GFP-positivo após transfecting a expressão I-SCEI plasmídeo. Esgotamento de BRCA1 reproducibly reduz a conversão de células positivas para GFP-por cerca de 8-10 vezes. Embora as porcentagens reais de GFP células positivas podem mudar de experimento para experimento, a proporção de células positivas para GFP-seguintes esgotamento BRCA1 em relação ao esgotamento de controle dentro de um experimento é bastante consistente. Nós normalizar os resultados, definindo a taxa de conversão desinibida a 100% e os resultados expressam com esgotamento BRCA1 e adicione-backs como porcentagens. Desta forma, podemos combinar múltiplas experiências, ea variação experimental não é alto. Adicionar volta de mutantes BRCA1, até agora, produziu resultados robustos: cada variante BRCA1 tem ou totalmente restaurado homóloga atividade recombinação ou não teve efeito. Uma consideração importante na obtenção destes resultados é que as nossas condições de transfecção produzir níveis de proteína BRCA1 que aproximadamente igual a concentração da proteína endógena nas células não tratadas. Muito alto de uma superexpressão de mutante mesmo um defeito de BRCA1 pode resultar em alguma atividade de recombinação homóloga positiva. Um problema que nós precisávamos de superar foi a toxicidade de transfecção. Esgotamento do BRCA1 em si pode retardar o crescimento de células, e do balanço de DNA Lipofectamine-2000 e plasmídeo em relação ao número de células no prato deve ser realizada em um nível consistente. DNA muito Lipofectamine ou plasmídeo irá reduzir o número de células eo número de células será insuficiente para obter resultados confiáveis ​​citometria de fluxo. O saldo dos reagentes plasmídeo e Lipofectamine versus o número de células transfectadas é muito importante para a manutenção da viabilidade das células. <img src="/files/ftp_upload/2468/2468fig1.jpg" alt="Figura 1" /> Figura 1. O substrato recombinação. A estratégia que tinha sido desenvolvido pelo grupo de M. Jasin 7,12 é retratado. O pDR GFP-plasmídeo contém dois alelos defeituosos da GFP, um dos quais contém um I-SCEI site de endonuclease de restrição. A linhagem de células HeLa derivados contém este substrato DNA integrados em um único site no genoma 9, e reparação ativa da quebra de DNA double-stranded no site I-SCEI por recombinação homóloga resultados na conversão de um alelo para GFP-positivo. <img src="/files/ftp_upload/2468/2468fig2.jpg" alt="Figura 2" /> Figura 2. Detecção de GFP células positivas por microscopia de fluorescência. As células foram testados de acordo com este protocolo e controle de depleção de células estavam ativas na recombinação homóloga, detectada por uma alta porcentagem de células com expressão GFP (esquerda). Esgotamento de BRCA1 por resultados RNAi em uma diminuição acentuada no número de células positivas para GFP-(direita). <img src="/files/ftp_upload/2468/2468fig3.jpg" alt="Figura 3" /> Figura 3. Resultados de um add-back de mutantes missense BRCA1. Os resultados de um único experimento são mostrados neste histograma. Na ausência de transfectadas I-SCEI expressar plasmídeo (pista 1) não há células GFP-positivos detectados. Os resultados em pistas 06/02 foram todas tomadas a partir de células em que o I-SCEI expressar plasmídeo foi transfectado no dia 3. Em células que foram perdidos por um gene irrelevante (luciferase) e com o vector add-back, 15,5% das células foram GFP-positivo (pista 2). Esgotamento dos genes BRCA1 e vector add-volta revela o efeito da perda de BRCA1 na recombinação homóloga (faixa 3). Os efeitos da depleção de BRCA1 e adicione-back de tipo selvagem BRCA1 (faixa 4), BRCA1 (M18T) (faixa 5), ​​e BRCA1 (I21V) (faixa 6) são mostrados. Estes resultados são de um único experimento, e seria combinada com pelo menos dois experimentos mais repetir a fim de obter uma medida da recombinação homóloga apoiado por uma proteína BRCA1 dado mutante.

<ol>
	<li>Carvalho, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+cancer+risk+associated+with+germ+line+BRCA1+missense+variants+by+functional+analysis.">Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis.</a> Cancer Res. 67, 1494-1501 (2007).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+epidemiology+of+BRCA1.">Genetic epidemiology of BRCA1.</a> Cancer Biol Ther. 3, 509-514 (2004).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+functional+effects+of+unclassified+genetic+variants.">Assessment of functional effects of unclassified genetic variants.</a> Hum Mutat. 29, 1314-1326 (2008).</li><li>Tomlinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+breast+cancer+cell+line+derived+from+a+germ-line+BRCA1+mutation+carrier.">Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier.</a> Cancer Res. 58, 3237-3242 (1998).</li><li>Ruffner, H., Joazeiro, C. A., Hemmati, D., Hunter, T., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-predisposing+mutations+within+the+RING+domain+of+BRCA1:+loss+of+ubiquitin+protein+ligase+activity+and+protection+from+radiation+hypersensitivity.">Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity.</a> Proc Natl Acad Sci U S A. 98, 5134-5139 (2001).</li><li>Moynahan, M. E., Chiu, J. W., Koller, B. H., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brca1+controls+homology-directed+DNA+repair.">Brca1 controls homology-directed DNA repair.</a> Mol Cell. 4, 511-518 (1999).</li><li>Nakanishi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Fanconi+anemia+monoubiquitination+pathway+promotes+homologous+DNA+repair.">Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.</a> Proc Natl Acad Sci U S A. 102, 1110-1115 (2005).</li><li>Pierce, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+breaks+and+tumorigenesis.">Double-strand breaks and tumorigenesis.</a> Trends Cell Biol. 11, 52-59 (2001).</li><li>Ransburgh, D. J., Chiba, N., Ishioka, C., Toland, A. E., Parvin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+breast+tumor+mutations+in+BRCA1+that+abolish+its+function+in+homologous+DNA+recombination.">Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination.</a> Cancer Res. 70, 988-995 (2010).</li><li>Easton, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+genetic+assessment+of+1,433+sequence+variants+of+unknown+clinical+significance+in+the+BRCA1+and+BRCA2+breast+cancer-predisposition+genes.">A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes.</a> Am J Hum Genet. 81, 873-883 (2007).</li><li>Snouwaert, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1+deficient+embryonic+stem+cells+display+a+decreased+homologous+recombination+frequency+and+an+increased+frequency+of+non-homologous+recombination+that+is+corrected+by+expression+of+a+brca1+transgene.">BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a brca1 transgene.</a> Oncogene. 18, 7900-7907 (1999).</li><li>Pierce, A. J., Hu, P., Han, M., Ellis, N., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ku+DNA+end-binding+protein+modulates+homologous+repair+of+double-strand+breaks+in+mammalian+cells.">Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells.</a> Genes Dev. 15, 3237-3242 (2001).</li><li>Spearman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinically+applicable+models+to+characterize+BRCA1+and+BRCA2+variants+of+uncertain+significance.">Clinically applicable models to characterize BRCA1 and BRCA2 variants of uncertain significance.</a> J Clin Oncol. 26, 5393-5400 (2008).</li><li>Sweet, K., Senter, L., Pilarski, R., Wei, L., Toland, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+BRCA1+ring+finger+variants+of+uncertain+significance.">Characterization of BRCA1 ring finger variants of uncertain significance.</a> Breast Cancer Res Treat. 119, 737-743 (2009).</li></ol>

Não há conflitos de interesse declarados.

A vantagem deste método é que podemos estudar a função da proteína BRCA1 na regulação do reparo do DNA por recombinação homóloga, utilizando células que tipo selvagem BRCA1 endogenamente expressa. Adotamos dois distintos métodos estabelecidos de estudar o processo de recombinação homóloga e da utilização de RNAi mediada por esgotamento para obter o novo método para testar variantes BRCA1 para a função no reparo do DNA. A chave para o sucesso deste método foi o de estabelecer uma linha celular prontamente transfectable, tais como HeLa, com o substrato de recombinação no genoma de tal forma que obtemos níveis muito elevados de conversão GFP. Tivemos vários clones selecionados da transformação original, a fim de obter um que não tinha nenhum sinal de GFP detectáveis ​​de fundo, mas em cima de transfecção da expressão I-SCEI plasmídeo teve um nível muito alto de conversão de GFP. Com este método, nós agora estamos investigando outros específicos BRCA1 resíduos de aminoácidos para a função na recombinação homóloga. Juntamente com os insights biológicos a partir deste trabalho, estas conclusões podem ser aplicadas a genética do câncer clínico. A elevada percentagem de mutações BRCA1 missense têm significado clínico desconhecido desde há muitas variantes raras e não há informação suficiente para muitas destas para determinar a associação do câncer através da análise de segregação genética 3,10,13,14. Usando este método, as consequências de uma variante específica sobre a função biológica do BRCA1 na regulação da recombinação homóloga poderia ser usado para informar as mulheres que carregam uma dessas variantes em seu genoma.

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identifying the effects of brca1 mutations on homologous recombination using cells that express endogenous wild-type brca1

A análise funcional de mutações missense pode ser complicada pela presença na célula da proteína endógena. Estrutura-função análises do BRCA1 foram complicados pela falta de um ensaio robusta para a proteína BRCA1 todo o comprimento e as dificuldades inerentes ao trabalho com linhagens de células que expressam a proteína BRCA1 hypomorphic 1,2,3,4,5. Nós desenvolvemos um sistema em que a proteína BRCA1 endógena em uma célula tinha plena esgotados por RNAi visando o 3&#39;-UTR do mRNA BRCA1 e substituído por co-transfecting um plasmídeo expressando uma variante BRCA1. Uma vantagem desse procedimento é que o silenciamento aguda de substituição BRCA1 e simultânea permitir que as células a crescer sem mutações secundárias ou adaptações que possam surgir ao longo do tempo para compensar a perda da função BRCA1. Este esgotamento e adicione-back procedimento foi realizado em uma linhagem de células HeLa-derivada que foi prontamente analisadas para a atividade de recombinação homóloga. O ensaio de recombinação homóloga é baseado em um método previamente publicado pelo qual um substrato de recombinação é integrado no genoma (Figura 1) 6,7,8,9. Este substrato recombinação tem a rara de corte I-SCEI site da enzima de restrição dentro de um alelo GFP inativos, ea jusante é um alelo GFP segundo inativos. Transfecção do plasmídeo que expressa I-SCEI resulta em uma quebra de cadeia dupla, que pode ser reparado por recombinação homóloga, e se a recombinação homóloga que reparar o quebrá-lo cria um alelo GFP ativo que é facilmente marcados por citometria de fluxo para expressão da proteína GFP . Esgotamento de BRCA1 endógena resultou em uma redução de 8-10 vezes na recombinação homóloga de atividade, e adicione-back de tipo selvagem função plasmídeo de recombinação homóloga totalmente restaurado. Quando os mutantes ponto específico de BRCA1 comprimento total foram expressos de co-transfectadas plasmídeos, o efeito do mutante missense específicos poderiam ser marcados. Como exemplo, a expressão da proteína BRCA1 (M18T), uma variante do significado clínico desconhecido 10, foi expresso nessas células, ele não conseguiu restaurar BRCA1-dependente de recombinação homóloga. Pela expressão de contraste, de outra variante, também de significado desconhecido, BRCA1 (I21V) totalmente restaurado BRCA1 dependentes de recombinação homóloga função. Esta estratégia de testar a função de mutações BRCA1 missense foi aplicado para outro sistema biológico assaying para a função centrossoma (Kais et al, observações não publicadas). No geral, esta abordagem é adequada para a análise de mutantes missense em qualquer gene que devem ser analisados ​​recessiva.

Watch this Scientific Journal Video about Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1 at JoVE.com

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Nós fornecemos um método para testar variantes BRCA1 em um ensaio de cultura de tecidos com base homóloga de reparação de danos no DNA de recombinação, esgotando proteína BRCA1 endógenos de uma célula usando RNAi e substituindo-o por um mutante ponto BRCA1 que contém uma mudança de codificação.

Identificar os efeitos das mutações BRCA1 em Recombinação Homóloga usando células que expressam BRCA1 tipo selvagem endógena

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

identifying-effects-brca1-mutations-on-homologous-recombination-using

Research

JoVE Journal

Biology

14.6K visualizações.  The Ohio State University. Nós fornecemos um método para testar variantes BRCA1 em um ensaio de cultura de tecidos com base homóloga de reparação de danos no DNA de recombinação, esgotando proteína BRCA1 endógenos de uma célula usando RNAi e substituindo-o por um mutante ponto BRCA1 que contém uma mudança de codificação.

Vídeo: Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Estudando os efeitos da rigidez Matrix em função celular usando acrilamida baseado Hidrogéis

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Biologia

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Analisando a função de GTPases Pequenas pela microinjeção de Plasmids em Polarized Células Epiteliais

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Um método de genotipagem baseados em PCR para distinguir entre variedades do tipo selvagem e Ornamental de Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombineering Construções recombinação homóloga em<em&gt; Drosophila</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Medição dos Efeitos de bactérias em<em&gt; C. Elegans</em&gt; Comportamento Usando um Retenção Ensaio Egg

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

gDNA Enriquecimento por uma tecnologia baseada em transposase para NGS Análise de toda a sequência de<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt; e 9 genes envolvidos na reparação de danos no DNA

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

A identificação da proteína-proteína Locais de interacção Usando Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Caracterização funcional de Na<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; Trocadores de intracelular compartimentos usando a Seleção Proton-matança de expressá-las na membrana plasmática

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Determinação de ácidos sialic em amostras de fígado e leite de tipo selvagem e<em&gt; CMAH</em&gt; Ratinhos de eliminação.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Um método para avaliar os efeitos da bacteriocina na microbiota intestinal de ratos

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...