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.

NGS 장치 및 기술의 광범위한 사용은 암과 유전 질환의 연구에 새로운 기회를 제공하고 있습니다. 전체 게놈 염기 서열 또는 RNA 서열에 더하여, 다수의 환자에서 선택된 서열의 gDNA를 다량의 분석을 동시에 진단에 큰 가능성을 제공한다. 여기에, 우리는 중간 서열 분석 장치 (자재 / 장비의 표)와 동시에 24 명에서 11 완전한 유전자를 연구하는 Nextera 기술을 사용하여 (필요시) 특정 디자인을 개발했다...

2010 년 (기본적으로 여성) 거의 1백50만명는 전 세계적으로 유방암을 개발했다. 그러나, 이들의 경우 5-10 %의 유전 있다고 추정된다. 유전성 유방암과 난소 암의 1에 관련된 거의 이십년 전, BRCA1과 BRCA2가 확인되었다. 약 15 년 전 이후로, BRCA1 및 BRCA2 코딩 영역은 유방암과 난소 암에 대한 유전 적 소인을 결정하는 순서가되었다. BRCA1 및 BRCA2의 변화는 이들 영역의 분석을 효과적으로 스크리닝 충분하지 않은 것을 시사 선택된 가족이 10-20 %가 검출된다. 최근, BRCA1 및 BRCA2의 비 - 코딩 서열 (프로모터, 인트론, 3 &#39;UTR)의 분석은 새로운 돌연변이 / 변형 유방암 3-6의 높은 위험에 링크 될 수 있다는 것을 강조 하였다. BRCA1과 BRCA2 단백질은 (상동 재조합 수리에 참여하고 있습니다수많은 파트너 (7)에 의해 완료 HHR). BRCA1 또는 BRCA2의 변화는 DNA 복구에 결함을 유발하지만, 다른 파트너는 유방암 위험에 영향을 미칠 수있다. 이 가설은 BRIP1 8 이후 확인 된 것으로 나타나고 PALB2 9는 각각 자궁 경부 암과 유방암에 대한 입증 영향을 미친다. 또한, 다른 두 &quot;중등도 위험&quot;유방암 감수성 유전자, ATM 및 CHEK2는 또한 정기적으로 열 연구 할 수있다. 이 연구에 이어, 우리는 동시에 상대적으로 매우 쉽고을 사용하여 24 명에서 11 유전자 (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAP80 및 TP53)를 분석 할 수있는 프로토콜을 개발하기로 결정 매체 처리 장치 및 농축 transposase 시퀀싱 기술을 기반으로 빠른 프로토콜. 감사합니다이 기술로, 우리는 2500 염기쌍의 인트론 영역이 포함되지 않은 RAP80, (: 176,381,588-176,390,180 CHR5)을 제외하고, 3&#39;-UTR의 단부에 프로모터 개시로부터 완전한 유전자 서열. 이는 2734 프로브 연구에 대한 1,000,300 염기쌍의 합계를 나타냅니다. 일반적으로 BRCA1과 BRCA2 엑손 서열은 20 명 미만 환자의 1.5 개월을 필요로 생거 시퀀싱에 의해 분석된다. 본 프로토콜 (도 1)와 동일한 시간에, 75 개 이상 11 명의 환자에 대한 완전한 유전자를 분석 할 수있다.

<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

차세대 시퀀싱의 광범위한 사용은 암 연구 및 진단을위한 새로운 길을 열었습니다. NGS는 거대한 암에 대한 새로운 데이터의 양, 특히 암 유전학을 가져올 것이다. 현재의 지식과 미래의 발견은 필요한 암의 유전 적 소인에 관여 할 수있는 유전자의 거대한 숫자를 연구 할 것입니다. 이러한 관점에서, 우리는 DNA 손상의 복구에 관여하는 유전자의 완전한 11 공부 Nextera 설계를 개발 하였다. 이 프로토콜은 안전하게 동시에 24 명에서 11 유전자 (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80 및 TP53) 프로모터에서 3&#39;-UTR을 연구하기 위해 개발되었다. transposase 기술과 gDNA를 농축에 따라이 프로토콜은, 다중화를 샘플링 할 수있는 유전자 진단 덕분에 시간의 측면에서 큰 장점을 제공합니다. 이 프로토콜은 혈액 gDNA를 안전하게 사용할 수있다.

샘플 QC 결과 표적 유전자의 서열을 결정하는 본 방법의 기능은 gDNA를 (도 2a)의 품질 및 tagmentation 단계의 품질에 기초한다. tagmentation은 (도 2b를 참조하면, 상부 패널) 충분하지 않은 경우, 시퀀스는 만족스럽지 않을 것이다. 전술 한 바와 같이, tagmentation 정제 후, gDNA를 약 300 bp의 단편의 대부분 (도 2B, 하부 패널)로 1000 염기쌍 150로부터 염기?...

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.

의 전체 순서의 NGS 분석을위한 Transposase 기반 기술에 의해 gDNA를 농축<em&gt; BRCA1</em&gt;<em&gt; BRCA2</emDNA 손상 복구에 참여&gt;, 9 유전자

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

12.1K 조회수.  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. 

비디오: 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.

조기 칙 태아의 현장 하이브리드화에서 더블 전체 산

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

연구

생물학

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.

이미징 불일치 복구 및 DNA의 손상 세포 응답 바실러스 subtilis</em

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

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.

형광 - 현미경 검사와 차세대 장면 : Organelle 무결성에 관련된 유전자의 식별을위한 유용한 도구

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

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Ribosome 바운드 Polypeptides을 파악하는 도구로 SecM 체포 시퀀스를 사용하여

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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.

선충류의 바디 크기 규정에 참여 유전자에 대한 화면으로 RNA로 인한 간섭 수유 전략을 사용하여<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.

이미징 세포 막 상해 및 수리에 관여하는 세포 내 프로세스

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

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

프로토 타입 소형 흐름 사이토와 동반자 미세 유체 혼합 기술의 감소 - 중력 환경 하드웨어 데모

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

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.

마우스 골격근에있는 검색, 탐지 DNA 손상 분석<em&gt; 현장에서</em&gt; TUNEL 방법을 사용하여

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.

면역형광에 의한 DNA 수리 단백질 상호작용의 시각화

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

An Integrated Approach for Microprotein Identification and Sequence Analysis

Next-generation sequencing (NGS) has propelled the field of genomics forward and produced whole genome sequences for numerous animal species and model organisms. However, despite this wealth of sequence information, comprehensive gene annotation efforts have proven challenging, especially for small proteins. Notably, conventional protein annotation methods were designed to intentionally exclude putative proteins encoded by short open reading frames (sORFs) less than 300 nucleotides in length to filter out the exponentially higher number of spurious noncoding sORFs throughout the genome. As a result, hundreds of functional small proteins called microproteins (&#60;100 amino acids in length) have been incorrectly classified as noncoding RNAs or overlooked entirely.
Here we provide a detailed protocol to leverage free, publicly available bioinformatic tools to query genomic regions for microprotein-coding potential based on evolutionary conservation. Specifically, we provide step-by-step instructions on how to examine sequence conservation and coding potential using Phylogenetic Codon Substitution Frequencies (PhyloCSF) on the user-friendly University of California Santa Cruz (UCSC) Genome Browser. Additionally, we detail steps to efficiently generate multiple species alignments of identified microprotein sequences to visualize amino acid sequence conservation and recommend resources to analyze microprotein characteristics, including predicted domain structures. These powerful tools can be used to help identify putative microprotein-coding sequences in noncanonical genomic regions or to rule out the presence of a conserved coding sequence with translational potential in a noncoding transcript of interest.

The protocol described here provides detailed instructions on how to analyze genomic regions of interest for microprotein-coding potential using PhyloCSF on the user-friendly UCSC Genome Browser. Additionally, several tools and resources are recommended to further investigate sequence characteristics of identified microproteins to gain insight into their putative functions.

마이크로단백질 동정 및 서열 분석을 위한 통합 접근법

Next-generation sequencing (NGS) has propelled the field of genomics forward and produced whole genome sequences for numerous animal species and model ...