We thank the Ligue contre le Cancer de C&#244;te d&#8217;Or and the Centre Georges-Fran&#231;ois Leclerc for their financial support. We thank Philip Bastable for the editing of the manuscript.

<ol>
	<li>Cornelisse, C. J., Cornelis, R. S., Devilee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+responsible+for+familial+breast+cancer.">Genes responsible for familial breast cancer.</a> Pathol. Res. Pract. 192 (7), 684-693 (1996).</li><li>Culver, J., Lowstuter, K., Bowling, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+breast+cancer+risk+and+BRCA1/2+carrier+probability.">Assessing breast cancer risk and BRCA1/2 carrier probability.</a> Breast Dis. 27, 5-20 (2007).</li><li>Cox, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+variants+of+the+BRCA1+wild-type+allele+modify+the+risk+of+breast+cancer+in+BRCA1+mutation+carriers.">Common variants of the BRCA1 wild-type allele modify the risk of breast cancer in BRCA1 mutation carriers.</a> Hum. Mol. Genet. 20 (23), 4732-4747 (2011).</li><li>Maia, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+BRCA2+cis-regulation+in+normal+breast+and+cancer+risk+amongst+BRCA2+mutation+carriers.">Effects of BRCA2 cis-regulation in normal breast and cancer risk amongst BRCA2 mutation carriers.</a> Breast Cancer Res. 14 (2), 63 (2012).</li><li>Anczuk&#243;w, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA2+deep+intronic+mutation+causing+activation+of+a+cryptic+exon:+opening+toward+a+new+preventive+therapeutic+strategy.">BRCA2 deep intronic mutation causing activation of a cryptic exon: opening toward a new preventive therapeutic strategy.</a> Clin. Cancer Res. 18 (18), 4903-4909 (2012).</li><li>Brewster, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+fifteen+novel+germline+variants+in+the+BRCA1+3'UTR+reveals+a+variant+in+a+breast+cancer+case+that+introduces+a+functional+miR-103+target+site.">Identification of fifteen novel germline variants in the BRCA1 3'UTR reveals a variant in a breast cancer case that introduces a functional miR-103 target site.</a> Hum. Mutat. 33 (12), 1665-1675 (2012).</li><li>Roy, R., Chun, J., Powell, S. 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+and+BRCA2:+different+roles+in+a+common+pathway+of+genome+protection.">BRCA1 and BRCA2: different roles in a common pathway of genome protection.</a> Nat Rev Cancer. 12 (1), 68-78 (2012).</li><li>Ma, X. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+evidence+for+the+contribution+of+the+genetic+variations+of+BRCA1-interacting+protein+1+(BRIP1)+to+the+genetic+susceptibility+of+cervical+cancer.">First evidence for the contribution of the genetic variations of BRCA1-interacting protein 1 (BRIP1) to the genetic susceptibility of cervical cancer.</a> Gene. 524 (2), 208-213 (2013).</li><li>Haanp&#228;&#228;, M., Pylk&#228;s, K., Moilanen, J. S., Winqvist, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+need+for+routine+clinical+testing+of+PALB2+c.1592delT+mutation+in+BRCA+negative+Northern+Finnish+breast+cancer+families.">Evaluation of the need for routine clinical testing of PALB2 c.1592delT mutation in BRCA negative Northern Finnish breast cancer families.</a> BMC Med. Genet. 14, 82 (2013).</li><li>Southey, M. C., Teo, Z. L., Winship, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PALB2+and+breast+cancer:+ready+for+clinical+translation.">PALB2 and breast cancer: ready for clinical translation.</a> Appl. Clin. Genet. 6, 43-52 (2013).</li><li>Ulahannan, D., Kovac, M. B., Mulholland, P. J., Cazier, J. B., Tomlinson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+and+implementation+issues+in+using+next-generation+sequencing+of+cancers+in+clinical+practice.">Technical and implementation issues in using next-generation sequencing of cancers in clinical practice.</a> Br. J. Cancer. 109, 827-835 (2013).</li><li>Hernan, I., Borr&#224;s, E., de Sousa Dias, M., Gamundi, M. J., Ma&#241;&#233;, B., Llort, G., Ag&#250;ndez, J. A., Blanca, M., Carballo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+genomic+variations+in+BRCA1+and+BRCA2+genes+by+long-range+PCR+and+next-generation+sequencing.">Detection of genomic variations in BRCA1 and BRCA2 genes by long-range PCR and next-generation sequencing.</a> J. Mol. Diagn. 14, 286-293 (2012).</li><li>Knierim, E., Lucke, B., Schwarz, J. M., Schuelke, M., Seelow, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+three+methods+for+fragmentation+of+long-range+PCR+products+for+next+generation+sequencing.">Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing.</a> Plos One. 6, e28240 (2011).</li><li>de Sousa Dias, M., Hernan, I., Pascual, B., Borr&#224;s, E., Ma&#241;&#233;, B., Gamundi, M. J., Carballo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+novel+mutations+that+cause+autosomal+dominant+retinitis+pigmentosa+in+candidate+genes+by+long-range+PCR+amplification+and+next-generation+sequencing.">Detection of novel mutations that cause autosomal dominant retinitis pigmentosa in candidate genes by long-range PCR amplification and next-generation sequencing.</a> Mol. Vis. 19, 654-664 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Breast+Cancer+Linkage+Consortium.+Cancer+Risks+in+BRCA2+Mutation+Carriers.">The Breast Cancer Linkage Consortium. Cancer Risks in BRCA2 Mutation Carriers.</a> J. Natl. Cancer Inst. 91, 1310-1316 (1999).</li><li>Levy-Lahad, E., Friedman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+risks+among+BRCA1+and+BRCA2+mutation+carriers.">Cancer risks among BRCA1 and BRCA2 mutation carriers.</a> Br. J. Cancer. 96 (1), 11-15 (2007).</li><li>Risch, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population+BRCA1+and+BRCA2+mutation+frequencies+and+cancer+penetrances:+a+kin-cohort+study+in+Ontario,+Canada.">Population BRCA1 and BRCA2 mutation frequencies and cancer penetrances: a kin-cohort study in Ontario, Canada.</a> J. Natl. Cancer Inst. 98 (23), 1694-1706 (2006).</li><li>Slater, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PALB2+mutations+in+European+familial+pancreatic+cancer+families.">PALB2 mutations in European familial pancreatic cancer families.</a> Clin. Genet. 78, 490-494 (2010).</li><li>Brennan, G. T., Relias, V., Saif, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA+and+pancreatic+cancer.">BRCA and pancreatic cancer.</a> J.O.P. 14 (4), 325-328 (2013).</li></ol>

The authors have no conflicts of interest to declare.

O amplo uso de dispositivos e tecnologias NGS tem proporcionado novas oportunidades no estudo de câncer e doenças genéticas. Além de seqüenciamento do genoma inteiro ou RNA sequenciamento, a análise de uma grande quantidade de seqüências gDNA selecionados em vários pacientes ao mesmo tempo oferece grandes perspectivas no diagnóstico. Aqui, desenvolvemos um projeto específico (disponível on demand) usando a tecnologia para estudar Nextera 11 genes completos em 24 pacientes, simultaneamente, com um dispositivo...

Em 2010, cerca de 1,5 milhões de pessoas (essencialmente mulheres) desenvolveram câncer de mama em todo o mundo. Estima-se que 5 a 10% dos casos eram hereditários. Quase 20 anos atrás, BRCA1 e BRCA2 foram identificados como envolvidos na mama hereditário e câncer de ovário 1. Desde cerca de 15 anos atrás, BRCA1 e BRCA2 regiões codificantes foram sequenciados para determinar a predisposição genética para câncer de mama e ovário. Alterações nos genes BRCA1 e BRCA2 são detectados em 10 a 20% das famílias seleccionadas 2 sugerindo que a análise destas regiões não é suficiente para um rastreio eficaz. Recentemente, a análise de seqüências não-codificantes (íntrons, promotores, 3&#39;UTR) de BRCA1 e BRCA2 destacou que novas mutações / variações poderiam estar ligadas a um maior risco de câncer de mama 3-6. Proteínas BRCA1 e BRCA2 estão envolvidas na reparação de recombinação homóloga (HHR), que é completado por inúmeros parceiros 7. Enquanto as alterações nos genes BRCA1 ou BRCA2 induzir defeitos no reparo do DNA, os outros parceiros também podem afetar o risco de câncer de mama. Esta hipótese parece ter sido validada desde BRIP1 8 e 9 PALB2 têm um impacto comprovado sobre o câncer do colo do útero e de mama, respectivamente. Além disso, dois outros genes &quot;risco moderado&quot; de câncer de mama susceptibilidade, ATM e CHEK2, também podem ser estudados rotineiramente 10. Na sequência destes estudos, decidimos desenvolver um protocolo para analisar 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAP80, e TP53) em 24 pacientes simultaneamente, utilizando uma forma muito fácil e relativamente protocolo rápido com base na tecnologia de transposase, com o enriquecimento e a sequenciação de um dispositivo de transferência médio. Obrigadoa esta técnica, foi sequenciado a partir de genes completos no início do promotor para a extremidade de 3&#39;-UTR, excepto para RAP80, para a qual não foi coberta uma região intrónica de 2500 pb (CHR5: 176,381,588-176,390,180). Isso representa um total de cerca de 1.000.300 bp estudou com 2.734 sondas. Normalmente, as sequências de BRCA1 e BRCA2 exônicos são analisados ​​por sequenciação de Sanger, que precisa de 1,5 meses para pacientes com menos de 20. Com o presente protocolo (Figura 1), ao mesmo tempo, 11 genes completos para mais de 75 pacientes poderia ser analisado.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MiSeq</td>
			<td>Illumina</td>
			<td>SY-410-1001</td>
			<td>Sequencing/medium throughput device</td>
		</tr>
		<tr>
			<td>Nextera Enrichment kit</td>
			<td>Illumina</td>
			<td>FC-123-1208</td>
			<td>Transposase based technology</td>
		</tr>
		<tr>
			<td>300 cycle cartridge</td>
			<td>Illumina</td>
			<td>15033624</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>Magnetic purification beads</td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Alpaqua</td>
			<td>A32782</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Life Technologies</td>
			<td>4306737</td>
			<td></td>
		</tr>
		<tr>
			<td>MIDI 96-well plates</td>
			<td>Biorad</td>
			<td>AB0859</td>
			<td></td>
		</tr>
		<tr>
			<td>Microseal A</td>
			<td>Biorad</td>
			<td>MSA-5001</td>
			<td>This seal is necessary only for PCR amplification. Other standard seals can be used throughout the experiment</td>
		</tr>
		<tr>
			<td>MiSeq</td>
			<td>Illumina</td>
			<td></td>
			<td>Provided with the sequencing device 
			Experiment Manager software</td>
		</tr>
		<tr>
			<td></td>
			<td>Illumina</td>
			<td></td>
			<td>Internet adress: http://designstudio.illumina.com/NexteraRc/project/new&#62; 
			Manufacturer website tool</td>
		</tr>
	</tbody>
</table>

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

O uso generalizado de Next Generation Sequencing abriu novos caminhos para a pesquisa do câncer e diagnóstico. NGS trará enormes quantidades de novos dados sobre o câncer e, especialmente, genética do câncer. O conhecimento atual e futuras descobertas tornará necessário estudar um grande número de genes que poderiam estar envolvidos em uma predisposição genética para o câncer. Neste sentido, foi desenvolvido um projeto para estudar Nextera 11 genes completos envolvidos no DNA reparação de danos. Este protocolo foi desenvolvido para estudar com segurança 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80 e TP53) do promotor de 3&#39;-UTR em 24 pacientes simultaneamente. Este protocolo, baseado em tecnologia de transposase e gDNA enriquecimento, dá uma grande vantagem em termos de tempo para o diagnóstico graças genética para provar multiplexação. Este protocolo pode ser utilizado com segurança com ADNg sangue.

Os resultados das amostras de CQ A capacidade do presente método para determinar sequências de genes alvo se baseia na qualidade do ADNg (Figura 2A) e a qualidade do passo tagmentation. Se o tagmentation não é suficiente (Figura 2B, painel superior), a sequenciação não será satisfatória. Como mencionado acima, após a purificação tagmentation, o ADNg deve ser tagmented em fragmentos de 150 pb e 1000 pb, com a maioria dos fragmentos de cerca de 300 pb...

Watch this Scientific Journal Video about 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 at JoVE.co

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 ...

gdna-enrichment-transposase-based-technology-for-ngs-analysis-whole

Research

JoVE Journal

Biology

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

12.2K visualizações.  Centre Georges-François Leclerc. 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. 

Vídeo: 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

Double Whole Mount in situ Hybridization of Early Chick Embryos

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  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Monte Whole dupla hibridização in situ de embriões de galinha precoce

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

Biologia

Method for Whole Mount Antibody Staining in Chick

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 antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Método para coloração Whole Mount anticorpos em Pintainho

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Mismatch Repair imagem e respostas celulares a danos no DNA em Bacillus subtilis</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

Análise de DNA dupla fita-Break Repair (DSB) em células de mamíferos

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Análise de Schwann-astrócito Interações Usando In Vitro Ensaios

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

Microscopia de fluorescência de Triagem e seqüenciamento da próxima geração: Ferramentas úteis para a identificação de genes envolvidos na integridade das Organelas

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Usando RNA mediada estratégia de alimentação Interferência em tela para genes envolvidos na regulação do tamanho corporal na Nemátodo<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

Imagens de células Lesão de Membrana e Processos Subcelulares envolvidas no reparo

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Detecção e análise de danos no DNA em Rato Músculo Esquelético<em&gt; In Situ</em&gt; Usando o método TUNEL

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.