We thank Erin Zoller, Jessica Bene, and Lindsey Hays for input and direction in protocol development. MTW was supported in part by NIH R21 HG008186 and a Trustee Award grant from the Cincinnati Children's Hospital Research Foundation. ZHP was supported in part by T32 GM063483-13.

<ol>
	<li>Hindorff, L. 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=Potential+etiologic+and+functional+implications+of+genome-wide+association+loci+for+human+diseases+and+traits.">Potential etiologic and functional implications of genome-wide association loci for human diseases and traits.</a> Proc Natl Acad Sci U S A. 106 (23), 9362-9367 (2009).</li><li>Maurano, M. 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=Systematic+localization+of+common+disease-associated+variation+in+regulatory+DNA.">Systematic localization of common disease-associated variation in regulatory DNA.</a> Science. 337 (6099), 1190-1195 (2012).</li><li>Ward, L. D., Kellis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+noncoding+genetic+variation+in+complex+traits+and+human+disease.">Interpreting noncoding genetic variation in complex traits and human disease.</a> Nat Biotechnol. 30 (11), 1095-1106 (2012).</li><li>Paul, D. S., Soranzo, N., Beck, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interpretation+of+non-coding+sequence+variation:+concepts+and+challenges.">Functional interpretation of non-coding sequence variation: concepts and challenges.</a> Bioessays. 36 (2), 191-199 (2014).</li><li>Zhang, F., Lupski, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-coding+genetic+variants+in+human+disease.">Non-coding genetic variants in human disease.</a> Hum Mol Genet. , (2015).</li><li>Lee, T. I., Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+and+its+misregulation+in+disease.">Transcriptional regulation and its misregulation in disease.</a> Cell. 152 (6), 1237-1251 (2013).</li><li>Slatkin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linkage+disequilibrium--understanding+the+evolutionary+past+and+mapping+the+medical+future.">Linkage disequilibrium--understanding the evolutionary past and mapping the medical future.</a> Nat Rev Genet. 9 (6), 477-485 (2008).</li><li>Bush, W. S., Moore, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11:+Genome-wide+association+studies.">Chapter 11: Genome-wide association studies.</a> PLoS Comput Biol. 8 (12), e1002822 (2012).</li><li>1000 Genomes Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+map+of+genetic+variation+from+1,092+human+genomes.">An integrated map of genetic variation from 1,092 human genomes.</a> Nature. 491 (7422), 56-65 (2012).</li><li>Chang, C. C., 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=Second-generation+PLINK:+rising+to+the+challenge+of+larger+and+richer+datasets.">Second-generation PLINK: rising to the challenge of larger and richer datasets.</a> Gigascience. 4, 7 (2015).</li><li>Purcell, S., 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=PLINK:+a+tool+set+for+whole-genome+association+and+population-based+linkage+analyses.">PLINK: a tool set for whole-genome association and population-based linkage analyses.</a> Am J Hum Genet. 81 (3), 559-575 (2007).</li><li>ENCODE Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Crawford, G. E., 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=Genome-wide+mapping+of+DNase+hypersensitive+sites+using+massively+parallel+signature+sequencing+(MPSS).">Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS).</a> Genome Res. 16 (1), 123-131 (2006).</li><li>Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y., Greenleaf, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposition+of+native+chromatin+for+fast+and+sensitive+epigenomic+profiling+of+open+chromatin,+DNA-binding+proteins+and+nucleosome+position.">Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position.</a> Nat Methods. 10 (12), 1213-1218 (2013).</li><li>Giresi, P. G., Kim, J., McDaniell, R. M., Iyer, V. R., Lieb, 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=FAIRE+Formaldehyde-Assisted+Isolation+of+Regulatory+Elements)+isolates+active+regulatory+elements+from+human+chromatin.">FAIRE Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin.</a> Genome Res. 17 (6), 877-885 (2007).</li><li>Kent, W. J., 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=The+human+genome+browser+at+UCSC.">The human genome browser at UCSC.</a> Genome Res. 12 (6), 996-1006 (2002).</li><li>Roadmap Epigenomics Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+111+reference+human+epigenomes.">Integrative analysis of 111 reference human epigenomes.</a> Nature. 518 (7539), 317-330 (2015).</li><li>Martens, J. H., Stunnenberg, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLUEPRINT:+mapping+human+blood+cell+epigenomes.">BLUEPRINT: mapping human blood cell epigenomes.</a> Haematologica. 98 (10), 1487-1489 (2013).</li><li>Liu, 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=Cistrome:+an+integrative+platform+for+transcriptional+regulation+studies.">Cistrome: an integrative platform for transcriptional regulation studies.</a> Genome Biol. 12 (8), R83 (2011).</li><li>Griffon, 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=Integrative+analysis+of+public+ChIP-seq+experiments+reveals+a+complex+multi-cell+regulatory+landscape.">Integrative analysis of public ChIP-seq experiments reveals a complex multi-cell regulatory landscape.</a> Nucleic Acids Res. 43 (4), e27 (2015).</li><li>Staudt, L. M., 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=A+lymphoid-specific+protein+binding+to+the+octamer+motif+of+immunoglobulin+genes.">A lymphoid-specific protein binding to the octamer motif of immunoglobulin genes.</a> Nature. 323 (6089), 640-643 (1986).</li><li>Singh, H., Sen, R., Baltimore, D., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nuclear+factor+that+binds+to+a+conserved+sequence+motif+in+transcriptional+control+elements+of+immunoglobulin+genes.">A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes.</a> Nature. 319 (6049), 154-158 (1986).</li><li>Weirauch, M. 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=Determination+and+inference+of+eukaryotic+transcription+factor+sequence+specificity.">Determination and inference of eukaryotic transcription factor sequence specificity.</a> Cell. 158 (6), 1431-1443 (2014).</li><li>Ward, L. D., Kellis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HaploReg:+a+resource+for+exploring+chromatin+states,+conservation,+and+regulatory+motif+alterations+within+sets+of+genetically+linked+variants.">HaploReg: a resource for exploring chromatin states, conservation, and regulatory motif alterations within sets of genetically linked variants.</a> Nucleic Acids Res. 40 (Database issue), D930-D934 (2012).</li><li>Boyle, A. 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=Annotation+of+functional+variation+in+personal+genomes+using+RegulomeDB.">Annotation of functional variation in personal genomes using RegulomeDB.</a> Genome Res. 22 (9), 1790-1797 (2012).</li><li>Hume, M. A., Barrera, L. A., Gisselbrecht, S. S., Bulyk, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniPROBE,+update+2015:+new+tools+and+content+for+the+online+database+of+protein-binding+microarray+data+on+protein-DNA+interactions.">UniPROBE, update 2015: new tools and content for the online database of protein-binding microarray data on protein-DNA interactions.</a> Nucleic Acids Res. 43 (Database issue), D117-D122 (2015).</li><li>Mathelier, 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=JASPAR+2014:+an+extensively+expanded+and+updated+open-access+database+of+transcription+factor+binding+profiles.">JASPAR 2014: an extensively expanded and updated open-access database of transcription factor binding profiles.</a> Nucleic Acids Res. 42 (Database issue), 142-147 (2014).</li><li>Smith, M. F., Delbary-Gossart, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+Mobility+Shift+Assay+(EMSA).">Electrophoretic Mobility Shift Assay (EMSA).</a> Methods Mol Med. 50, 249-257 (2001).</li><li>Franza, B. R., Josephs, S. F., Gilman, M. Z., Ryan, W., Clarkson, B. <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+cellular+proteins+recognizing+the+HIV+enhancer+using+a+microscale+DNA-affinity+precipitation+assay.">Characterization of cellular proteins recognizing the HIV enhancer using a microscale DNA-affinity precipitation assay.</a> Nature. 330 (6146), 391-395 (1987).</li><li>. BCA Protein Assay Kit: User Guide Available from: <a target="_blank" href="https://tools.thermofisher.com/content/sfs/manuals/MAN0011430_Pierce_BCA_Protein_Asy_UG.pdf">https://tools.thermofisher.com/content/sfs/manuals/MAN0011430_Pierce_BCA_Protein_Asy_UG.pdf</a> (2014)</li><li>Wijeratne, A. B., 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=Phosphopeptide+separation+using+radially+aligned+titania+nanotubes+on+titanium+wire.">Phosphopeptide separation using radially aligned titania nanotubes on titanium wire.</a> ACS Appl Mater Interfaces. 7 (21), 11155-11164 (2015).</li><li>Silva, J. M., McMahon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fastest+Western+in+Town:+A+Contemporary+Twist+on+the+Classic+Western+Blot+Analysis.">The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis.</a> J. Vis. Exp. (84), (2014).</li><li>Lu, X., 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=Lupus+Risk+Variant+Increases+pSTAT1+Binding+and+Decreases+ETS1+Expression.">Lupus Risk Variant Increases pSTAT1 Binding and Decreases ETS1 Expression.</a> Am J Hum Genet. 96 (5), 731-739 (2015).</li><li>Ramana, C. V., Chatterjee-Kishore, M., Nguyen, H., Stark, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+roles+of+Stat1+in+regulating+gene+expression.">Complex roles of Stat1 in regulating gene expression.</a> Oncogene. 19 (21), 2619-2627 (2000).</li><li>Fillebeen, C., Wilkinson, N., Pantopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+Mobility+Shift+Assay+(EMSA)+for+the+Study+of+RNA-Protein+Interactions:+The+IRE/IRP+Example.">Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example.</a> J. Vis. Exp. (94), e52230 (2014).</li><li>Heng, T. S., Painter, 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=Immunological+Genome+Project,+C.+The+Immunological+Genome+Project:+networks+of+gene+expression+in+immune+cells.">Immunological Genome Project, C. The Immunological Genome Project: networks of gene expression in immune cells.</a> Nat Immunol. 9 (10), 1091-1094 (2008).</li><li>Wu, C., 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=BioGPS:+an+extensible+and+customizable+portal+for+querying+and+organizing+gene+annotation+resources.">BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources.</a> Genome Biol. 10 (11), R130 (2009).</li><li>Wu, C., Macleod, I., Su, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BioGPS+and+MyGene.info:+organizing+online,+gene-centric+information.">BioGPS and MyGene.info: organizing online, gene-centric information.</a> Nucleic Acids Res. 41 (Database issue), D561-D565 (2013).</li><li>Wang, J., 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=Sequence+features+and+chromatin+structure+around+the+genomic+regions+bound+by+119+human+transcription+factors.">Sequence features and chromatin structure around the genomic regions bound by 119 human transcription factors.</a> Genome Res. 22 (9), 1798-1812 (2012).</li><li>Holden, N. S., Tacon, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+problems+of+the+electrophoretic+mobility+shift+assay.">Principles and problems of the electrophoretic mobility shift assay.</a> J Pharmacol Toxicol Methods. 63 (1), 7-14 (2011).</li><li>Xu, J., Liu, H., Park, J. S., Lan, Y., Jiang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osr1+acts+downstream+of+and+interacts+synergistically+with+Six2+to+maintain+nephron+progenitor+cells+during+kidney+organogenesis.">Osr1 acts downstream of and interacts synergistically with Six2 to maintain nephron progenitor cells during kidney organogenesis.</a> Development. 141 (7), 1442-1452 (2014).</li><li>Yang, T. -. 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=Genevar:+a+database+and+Java+application+for+the+analysis+and+visualization+of+SNP-gene+associations+in+eQTL+studies.">Genevar: a database and Java application for the analysis and visualization of SNP-gene associations in eQTL studies.</a> Bioinformatics. 26 (19), 2474-2476 (2010).</li><li>Fort, 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=A+liver+enhancer+in+the+fibrinogen+gene+cluster.">A liver enhancer in the fibrinogen gene cluster.</a> Blood. 117 (1), 276-282 (2011).</li><li>Solberg, N., Krauss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luciferase+assay+to+study+the+activity+of+a+cloned+promoter+DNA+fragment.">Luciferase assay to study the activity of a cloned promoter DNA fragment.</a> Methods Mol Biol. 977, 65-78 (2013).</li><li>Rahman, M., 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=A+repressor+element+in+the+5'-untranslated+region+of+human+Pax5+exon+1A.">A repressor element in the 5'-untranslated region of human Pax5 exon 1A.</a> Gene. 263 (1-2), 59-66 (2001).</li><li>Mali, 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=RNA-Guided+Human+Genome+Engineering+via+Cas9.">RNA-Guided Human Genome Engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li></ol>

著者らは、開示することは何もありません。

<html>シーケンシングおよびジェノタイピング技術の進歩は著しく、疾患に関連する遺伝子変異体を同定するために我々の能力を強化しているが、これらの変異体によって影響を受ける機能のメカニズムを理解するために我々の能力が遅れています。問題の主な原因は、多くの疾患関連変異体は、上のコード可能性が高い遺伝子発現を制御困難ツー予測メカニズムに影響を与えるゲノムの領域、n個に位置していることです。ここでは、EMSAとDAPA技術に基づくプロトコル、おそらく多くの非コード変異体の機能に寄与遺伝子型に依存するTF結合事象を識別するための貴重な分子ツールを提示します。これらの二つの技術は、過去に広く使用されてきたが、それらは、ごく最近TF結合の遺伝的変異の分析のために適応されています。 TFを超えて、EMSAは、プロトコル36にわずかな調整をRNA結合タンパク質の遺伝的変異の効果を分析するために使用することができます。 ove_contentは &quot;&gt;本明細書に提示プロトコルは、シンプルかつ実行するのは簡単です。それにもかかわらず、特定の部分は、実験前に追加の考慮を必要とする第一に、厳密な統計分析を行うことで、検討のための変異体の最初のリストを生成することが重要であるこのステップでエラー。多くの変異体が疾患リスクに寄与することなく、核ライセートを差動的に結合することができるよう、すべてのダウンストリーム解析を脱線させることができます。また、適切な細胞型で行われる機能ゲノミクスデータの使用が重要である、などの無関係な細胞型は結合偽陽性TFの発生につながる可能性があり現在、最も広く使用されている機能ゲノムリソースはENCODE 12とロードマップエピゲノミクス17を含む。予測。追加情報興味のある疾患に関連する細胞型における遺伝子発現レベルに関しては、そのようなImmGen 37、BioGPS 38などの他の資源から得ることができます39、及びSNPsea 20。例えば、resourCESは、関連する細胞型で発現されるもののTFを含むだけにTF結合予測をフィルタリングするために使用することができます。 このようなEMSAやDAPAなどのin vitro実験の注意事項を考慮することも重要です。特に、偽陰性を低減するために別個の核溶解物調製物を用いて実験を繰り返す必要があります。また、成功したEMSA-スーパーシフトは、抗体ブロックTFのDNA結合ドメイン、抗体がTFに結合したEMSAバンドのさらなるシフト、またはバンドの損失、として提示することができます。いずれのシナリオでは、アイソタイプ対照抗体および/または異なるTFに向けた抗体を含むことはスーパーシフトの特異性を確認するのに有用です。スーパーシフトを行う際のもう1つの考慮事項は、反応にDTT /ポリソルベートを含めるかどうかです。 DTT /ポリソルベートは、結合していないDNAのより正確な定量を可能にローディング色素を安定化させます。しかしながら、それはまた、抗体のジスルフィド結合を減少させることができる結果EMSA-スーパーシフトの故障でる。複合体をスーパーシフトしようとしたときにDTT /ポリソルベートととせずに反応を試すことをお勧めします。ポリD（IC）と反応当たりサケ精子DNAの最適量は、滴定により実験的に決定されなければなりません。一般的には、1-6μgのポリD（IC）および50-500 ngのサケ精子の範囲を滴定することで十分です。 DAPAするための1つの有用なポジティブコントロールは、（CIS-BP 23またはFactorbook 40などのデータベースから取得した）十分に特徴付け結合モチーフとTFのためのコンセンサス配列を使用して、そのTFに特異的な抗体を用いたウェスタンを実行する必要があります。両方のアッセイのために、スクランブルされたオリゴは、任意の目的のオリゴに特異的である結合を観察することを示すために使用することができます。 どちらもEMSAとDAPA技術は実験的な制限があります。例えば、TFとDNAとの結合親和性は、緩衝液条件によって大部分は影響を受けています。理想的には、緩衝液条件は、内因性conditioを模倣する必要があります最適な結合を可能にするために、核のナノ秒。 EMSAのために、不適切なバッファー条件は、弱いバンドまたは完全にバンドが失われる可能性があります。 DAPA、非理想的な状態は、TF（s）は、洗浄工程中に溶出させることができます。したがって、各アッセイは、特定のバッファー条件の下で結合TF-オリゴを識別する際にのみ有効です。最も普遍的に有用な緩衝液条件は、上記のプロトコールに示されています。第2の制限は、EMSAとDAPA法からの実験結果はのTFは、オリゴに結合し、それを通してメカニズムについてはほとんど情報を提供することです。 TFは、オリゴに直接結合するか、またはその他の要因によって動員さ。従って、のTFをオリゴに結合し、実験的に、これらの予測を確認する方法を予測する計算オリゴ配列を分析することが重要です。例えば、特異的結合配列を変異させることができる、または結合パートナーは、実験的に枯渇させることができます。最後に、使用される抽出物の量は、各実験のtについて滴定される必要がありますO最適な結果を取得します。あまりにも多くの溶解物を結合TF-オリゴを飽和させ、リスクと非危険対立遺伝子間の結合の任意の差動を曖昧にすることができます。追加のトラブルシューティングのために、読者はいくつかの優れたレビューと方法原稿30,41,42を参照することができます。 ここに記載のアッセイに加えて、さらに、生細胞内の変異体（ 図1、下）の役割を研究するために利用可能なインビボアッセイには様々なものがあります。クロマチン免疫沈降は、 インビトロアッセイ内で観察された結合差がセル34生体内で複製される場合、研究が識別するために行うことができる対立遺伝子特異的な定量的ポリメラーゼ連鎖反応（チップ定量PCR）を行いました。関心対象の変異についてヘテロ接合性の細胞を使用する場合、例えば、定量PCR実験は、TF免疫沈降クロマチン内の2つの対立遺伝子間の濃縮の差を検出することができます。チップは、特定のChIPグレード抗体を必要とします。 howeveChIPグレードTF抗体が使用できない場合、R、フラッグタグ付きTFを用いて細胞をトランスフェクトすると、効果的な代替43です。さらに、標的遺伝子の発現に示差的結合の効果は、そのようなGenevar 44によってコンパイルされたもののような遺伝子型を特定の細胞または公的に利用可能な資源からローカルに収集した発現データを使用して、関連する細胞型における発現の量的形質遺伝子座（eQTL）分析を通じて調査することができます。ルシフェラーゼレポーターアッセイは、タンパク質の異なる結合程度を探索するためにも使用することができる遺伝子発現に影響を​​与えます。このようなアッセイにかかわらず変異体は、プロモーター、エンハンサー、リプレッサーまたは領域45-47に位置しているかどうかに動作するように修正することができます。最後に、CRISPR / Cas9 48としてゲノム編集技術は、因果関係を確認するために不可欠である単一の変異体でのみ異なる細胞株を作製するために使用することができます。このような技術は、実質的にbetw観察実験の分散を減らすことができます遺伝子発現または他の疾患の中間表現型を分析する機能読み出しが編集された細胞株で行い、非編集された細胞株と比較することができるので、EEN細胞株は、遺伝的に多様な被験体に由来します。 提示戦略の主な利点は、それが結合する遺伝子型依存性TFの容易かつ迅速な検出を可能にすることです。重要な遺伝的変異を優先することにより、更なる実験は、それらの生物学的効果を識別し、それらの因果関係を実証するために設計することができます。注目すべきことに、このプロトコルは、GWASまたはファインマッピングから特定された疾患または表現型関連バリアントを調査するために適用することができます。統計的に関連する遺伝的変異の遺伝子データとリストの大規模、成長宝庫はすでに利用可能です。ほとんどの場合、これらの変異体の統計的会合を駆動する生物学的メカニズムは明らかではありません。この提案に概説戦略は、多くの非コードdiseaの正確な機能的な解釈を可能にしますSE-関連バリアント。このような知識は、任意の遺伝子ベースの疾患を駆動する分子メカニズムの完全解明に不可欠です。

ゲノムワイド関連研究（GWAS）を含むシーケンシングおよびジェノタイピングベースの研究、候補遺伝子座の研究、およびディープシーケンシング研究は、統計学的疾患、形質または表現型と関連している多くの遺伝的変異を同定しました。初期の予測に反して、これらの変異体（85-93％）の大部分は非コード領域に位置しており、タンパク質1,2のアミノ酸配列を変更しません。これらの非コード変異体の機能の解釈と関連する疾患、形質または表現型にそれらを接続する生物学的メカニズムを決定することは3-6に挑戦証明されています。遺伝子発現 - 私たちは、重要な中間表現型にバリアントをリンクする分子メカニズムを識別するための一般的な戦略を開発しました。このパイプラインは、特に遺伝的変異による結合TFの調節を同定するために設計されています。この戦略を予測することを目的とした計算手法および分子生物学技術を組み合わせインシリコ候補変異体の生物学的効果、および（ 図1）、経験的に、これらの予測を検証します。 <img alt="図1" src="/files/ftp_upload/54093/54093fig1.jpg" /> 図1： 非コード遺伝子変異体の解析のための戦略的アプローチ 、この原稿に関連付けられた詳細なプロトコールに含まれていない手順は灰色で網掛けされている。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54093/54093fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 多くの場合、それぞれの統計的に関連する変異体と高い連鎖、不平衡（LD）にあるすべてのものを含むように変異体のリストを拡張することによって開始することが重要です。 LDは、R 2統計7によって測定することができる2つの異なる染色体位置における対立遺伝子の非ランダムな関連の尺度です。 r 2は林の尺度でありますR 2の変種間の完全な結合を示す= 1 2を持つ2つの変異体、間の影の不均衡。高LDにおける対立遺伝子は祖先集団全体の染色体上に同時分離することが判明しています。現在のジェノタイピングアレイは、ヒトゲノム中のすべての既知の亜種が含まれていません。その代わりに、彼らは人間のゲノム内のLDを利用し、LD 8の特定の領域内の他の変異体のためのプロキシとして機能する知られている変異体のサブセットを含みます。それは意味のある生物学的効果との因果バリアントバリアントとLDにあるため、このように、任意の生物学的影響のない変異体は、特定の疾患に関連することができます。手続きは、PLINK 10,11、全ゲノム関連解析のためのオープンソースのツールと互換性のあるバイナリファイルに千ゲノムプロジェクト9バリアントコールファイル（VCF）の最新リリースを変換することをお勧めします。続いて、各入力遺伝VAとLDのR 2&gt; 0.8と他のすべての遺伝的変異ほほ笑むは候補として同定することができます。変異体がヨーロッパ系の科目で同定された場合、同様の祖先の被験者からのデータは、LDの拡張のために使用されるべきで、STEP- 例えばこのための適切な参照集団を使用することが重要です。 LDの拡張はしばしば、候補変異体の数十をもたらし、これらのうちのごく一部が、疾患機構に寄与する可能性があります。多くの場合、実験的に個別にこれらの変異体のそれぞれを調べることは実行不可能です。バリアントの優先順位を決定するためのフィルタとして一般に公開機能ゲノムデータセットの数千人を活用することが有効です。例えば、ENCODEコンソーシアム12は 、DNアーゼ-seqの13、ATACなどの技術からクロマチンアクセシビリティデータと共に、文脈の広い範囲でのTFおよび補因子の結合を記述したChIP-seqの実験で何千もの、およびヒストンマークを行いました-seq 14、およびFAIRE-seqの15。 DATABこのようなUCSCゲノムブラウザ16、ロードマップエピゲノミクス17、青写真エピゲノム18、Cistrome 19、及び再マップ20としてのASおよびWebサーバは、細胞型および条件の広い範囲にわたって、これらおよび他の実験技術によって生成されたデータへの無料アクセスを提供します。実験的に調べるためにあまりにも多くの変異体が存在する場合、これらのデータは、関連する細胞および組織型における可能性の調節領域内に位置するものを優先順位付けするために使用することができます。さらに、変異体​​は、特定のタンパク質のためのChIP-seqのピークの範囲内にある場合には、これらのデータは、特定のTF（複数可）またはその影響を与える可能性があります結合補因子に関して潜在的なリードを提供することができます。 次に、優先順位を付け、得られた変異体は、EMSA 21,22を使用して予測結合遺伝子型依存性タンパク質を検証するために実験的にスクリーニングされます。 EMSAは、非還元TBEゲル上でオリゴの移行の変化を測定します。蛍光標識オリゴとともにインキュベートします核溶解液、および核因子の結合は、ゲル上のオリゴの動きを遅らせるます。このように、より多くの核因子を結合した​​オリゴは、スキャン時に強い蛍光シグナルとして提示します。特に、EMSAは、バインディング影響される特定のタンパク質についての予測を必要としません。 変異体は、予測調節領域内に位置し、示差的結合核因子することができるされていることが確認されると、計算方法は、その結合それらが影響する可能性のある特定のTF（複数可）を予測するために使用されます。私たちは、CIS-BP 23,24、RegulomeDB 25、UniProbe 26、およびJASPAR 27を使用することを好みます。候補者のTFが識別されると、これらの予測は、具体的には、これらのTF（EMSA-スーパーシフトとDAPA-西部劇）に対する抗体を用いて試験することができます。 EMSA-スーパーシフトは、核溶解液とオリゴにTF特異的抗体の添加を含みます。 EMSA-スーパーシフトで陽性の結果がのreprですEMSAバンドのさらなるシフト、またはバンドの損失としてesented（参照28に概説されています）。相補DAPAにおいて、変異体および20塩基対の隣接ヌクレオチドを​​含む5 &#39;ビオチン化オリゴ二本鎖を特異的オリゴを結合、核因子を捕捉するために、関連する細胞型（複数可）からの核溶解物と共にインキュベートします。オリゴ二重核因子複合体を磁気カラムにビーズをストレプトアビジンにより固定されています。バインドされた核要因が溶出29,48を介して直接収集されます。結合予測は、その後、タンパク質に特異的な抗体を用いたウェスタンブロットによって評価することができます。明らかな予測、またはあまりにも多くの予測が存在しない場合には、DAPA実験の変異プルダウンからの溶出は、質量分析を使用して候補のTFを同定するためにプロテオミクスコアに送信することができ、続いて前述のこれらを使用して検証することができますメソッド。 articlの残りの部分でE、EMSA及び遺伝的変異のDAPA分析のための詳細なプロトコルが提供されます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Custom DNA Oligonucleotides</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>http://www.idtdna.com/site/order/oligoentry</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td>KCl, for CE buffer</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Fisher Scientific</td>
			<td>15630-080</td>
			<td>For CE and NE buffer</td>
		</tr>
		<tr>
			<td>EDTA (0.5M), pH 8.0</td>
			<td>Life Technologies</td>
			<td>R1021</td>
			<td>For CE, NE, and annealing buffer</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td>NaCl, for NE buffer</td>
		</tr>
		<tr>
			<td>Tris-HCl (1M), pH 8.0</td>
			<td>Invitrogen</td>
			<td>BP1756-100</td>
			<td>For annealing buffer</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1X)</td>
			<td>Fisher Scientific</td>
			<td>MT21040CM</td>
			<td>PBS, for cell wash</td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol solution (1M)</td>
			<td>Sigma</td>
			<td>646563</td>
			<td>Reducing agent</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Thermo Scientific</td>
			<td>36978</td>
			<td>Protease Inhibitor</td>
		</tr>
		<tr>
			<td>Phosphatase Inhibitor Cocktail&#160;</td>
			<td>Thermo Scientific</td>
			<td>78420</td>
			<td>Prevents dephosphorylation of TFs</td>
		</tr>
		<tr>
			<td>Nonidet P-40 Substitute</td>
			<td>IBI Scientific</td>
			<td>IB01140</td>
			<td>NP-40, for nuclear extraction</td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>For measuring protein concentration</td>
		</tr>
		<tr>
			<td>Odyssey EMSA Buffer Kit</td>
			<td>Licor</td>
			<td>829-07910</td>
			<td>Contains all necessary EMSA buffers</td>
		</tr>
		<tr>
			<td>TBE Gels, 6%, 12 Wells</td>
			<td>Invitrogen</td>
			<td>EC6265BOX</td>
			<td>For EMSA</td>
		</tr>
		<tr>
			<td>TBE Buffer (10X)</td>
			<td>Thermo Scientific</td>
			<td>B52</td>
			<td>For EMSA</td>
		</tr>
		<tr>
			<td>FactorFinder Starting Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-318</td>
			<td>Contains all necessary DAPA buffers</td>
		</tr>
		<tr>
			<td>Licor Odyssey CLx</td>
			<td>Licor</td>
			<td></td>
			<td>Recommended scanner for DAPA/EMSA</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>Contains 10,000 units/mL of penicillin, 10,000 &#181;g/mL of streptomycin, and 25 &#181;g/mL of Fungizone&#174; Antimycotic</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td>FBS, for culture media</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Gibco</td>
			<td>22400-071</td>
			<td>Contains L-glutamine and 25mM HEPES</td>
		</tr>
	</tbody>
</table>

screening for functional non-coding genetic variants using electrophoretic mobility shift assay (emsa) and dna-affinity precipitation assay (dapa)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

このセクションでは、EMSAまたはDAPAを実行するときに何を期待するの代表的な結果が提供され、そして溶解物の品質に関して変動性が特徴です。例えば、複数回の凍結融解のタンパク質試料は変性をもたらし得ることが示唆されています。これらの「凍結融解」サイクルの文脈においてEMSA分析の再現性を調査するために、1遺伝子変異体で異なる2 35 bpのオリゴが指示された量のために解凍し、再凍結した核溶解物の単一バッチと共にインキュベートしました。回の図2を 、この特定のBリンパ球の核抽出液まで5回凍結融解することは、一見するタンパク質の完全性に影響を与えないことを示しています。しかし、核タンパク質の安定性は、サンプルによって異なりますので、使用する個々のセルラインでテストする必要があります。異なるバッチpreparからの変化を有することも可能です核溶解物のations。この変化は溶解前に、細胞継代数、または他の要因に細胞周期の段階によるものであるかどうかは、実際の結果を確実にするために、溶解物の異なるバッチを使用して、EMSAの結果を再現することが重要です。 加えて、EMSAのための信号対雑音比を最適化することが重要です。この中で重要な変数は、オリゴ濃度です。オリゴ（5-300フェムトモル）の量は、オリゴの異なる量が信号（ 図3）にどのように影響するかを評価するために滴定しました。アップオリゴ100フェムトモルのバンドの強度の増加が観察されました。この後、100フェムトモルを示唆するシグナルプラトーこのEMSA反応のための最適なオリゴ量です。 最後に、この原稿に記載の戦略の一部を使用して、当社の公表された研究の一つの代表的な図は、（ 図4）が設けられています。目で34の研究で、我々は、rs6590330増加のループス関連リスクアレルはSTAT1、下流の1型IFN受容体複合体35の遺伝子発現の相乗的な活性化および阻害の両方に関与する転写因子が結合することを示しました。この例では、STAT1は、第一のタンデム質量分析（DAPA-MS）に連結された高速液体クロマトグラフィーを使用してプロテオーム解析に続いDAPAにより同定しました。 DAPA-ウエスタンブロットは、プロテオーム解析（STAT1）から同定されたTFを確認し、STAT1のリン酸化形態は、非参照（ループスリスク）オリゴに結合したことを確認するために使用されました。この図は、この戦略の種々のアッセイが、非コーディング変異体転写因子の結合差を同定することができる方法を示します。 <img alt="図2" src="/files/ftp_upload/54093/54093fig2.jpg" /> 図2： 再現性の分析そして、EMSAの結果についての凍結融解サイクルの結果は。遺伝子変異体の参照（R）または非基準（NR）対立遺伝子を含むオリゴは、凍結融解の複数サイクル後の溶解物の同じ準備をプローブするために使用しました。 （リジン：溶解物）。各レーンは、ゲル（青い矢印）の下部にある無料のプローブが含まれています。オリゴにTFの結合は、ゲル（赤矢印）の上半分にバンドとして見ることができます。ゲル（ブラケットを参照）の下半分内に含まれるバンドは非特異的である。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54093/54093fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/54093/54093fig3.jpg" /> 図 3： 異なるオリゴ濃度の効果の評価オリゴの種々の濃度の核溶解物の単一製剤をプローブするために使用しました。 Fluorescent信号オリゴ制限試薬であることを示す、増加した量のオリゴと共に増加します。タンパク質の量は限定試薬となり、100〜300 fmolのレーンの信号プラトーが、。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54093/54093fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/54093/54093fig4.jpg" /> 図4：rs6590330 のループスリスクアレルは、DAPA-MS STAT1と高い非リスク対立遺伝子と比較して、rs6590330のリスク対立遺伝子を含むオリゴに結合pSTAT1の展示によって評価されるようSTAT1は、結合増加します 。ビオチン標識オリゴをエプスタイン - バーウイルス形質転換B細胞の核抽出物とインキュベートしました。オリゴに結合したタンパク質は、DAPAを使用して捕捉しました。タンパク質をSDSポリアクリルアミドゲル電気泳動により分離し、抗pSTAT1を用いて検出しました。（A）または抗STAT1（B）。 M：マーカー。 NR：rs6590330の非リスクアレルを含むオリゴ; R：rs6590330のリスク対立遺伝子を含むオリゴ;ミュータント：rs6590330の下流破砕推定されるSTAT1結合部位を含むオリゴ;細胞溶解物：B細胞からの核抽出物。バンドの相対強度は、各バンドの下に示されています。結果は、4つの実験の代表です。すべての実験は、STAT1は、リスク対立遺伝子を有するプローブへの結合の増加実証しながら、両方が危険オリゴから免疫沈降物中で検出されたが、2月4日の実験にはSTAT1またはpSTAT1は、非危険オリゴから免疫沈降物中に検出されませんでした。この図は、基準34から変更されている。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54093/54093fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA at JoVE.com

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

機能電気泳動移動度シフトアッセイ（EMSA）を用いた非コーディング遺伝的変異体およびDNA親和性沈殿アッセイ（DAPA）のスクリーニング

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...

screening-for-functional-non-coding-genetic-variants-using

Nanyang Technological University

Research

JoVE Journal

Biology

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

12.9K ビュー.  Cincinnati Children's Hospital. We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

ビデオ: Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

Light/dark Transition Test for Mice

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

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

マウス用ダーク/ライト遷移テスト

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

生物学

Assay for Neural Induction in the Chick Embryo

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

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

鶏胚における神経誘導のためのアッセイ

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

Elevated Plus Maze for Mice

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


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

マウス用高架式十字迷路

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

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

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

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

成体マウスから海馬歯状回の解剖

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

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

脂質中間相を用いた構造決定のための膜タンパク質の結晶化

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

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

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

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

遺伝的関連の機能的基準をテストするための対立遺伝子特異的遺伝子発現アッセイ

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

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

GIRKチャネルのモジュレーターを同定するための蛍光スクリーニングアッセイ

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

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

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

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

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

細菌機能ネットワークとPathwaysでのマッピング<em&gt;大腸菌</em&gt;合成遺伝子配列を用いて

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

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

自家製デュアルグロールシフェラーゼアッセイを用いて、ハイスループット機能スクリーニング

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

IRE / IRP例：RNA  - タンパク質相互作用の研究のための電気泳動移動度シフトアッセイ（EMSA）

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...