نشكر الحياة سيجما كامل العلم بعثة الهدف رقم مكتبة فريق التطوير، خصوصا كيفن Gutshall من أجل العثور على التكنولوجيا والتفاوض على شروط الترخيص، وHolemon هيذر لتقديم الدعم لها والمشاركة في مناقشات مثمرة استكشاف الأخطاء وإصلاحها. كما نشكر الدكتور يوب Gäken من كينجز كوليدج بلندن لتبادل بحرية النتائج غير المنشورة والاقتراحات، وقاضي حامد RxBiosciences لتحضير وجبات كبيرة من [كدنا]، واصراره في الحصول على أنها مستنسخة. نود أن نعترف نان لين وبحر سكوت من SAFC لتنفيذ المصفوفات [كدنا] وتحليل البيانات. المهمة هي علامة تجارية مسجلة لشركة التكنولوجيا الحيوية سيغما الدريخ

<ol>
	<li>Huang, Q., Gumireddy, K., Schrier, 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+microRNAs+miR-373+and+miR-520c+promote+tumour+invasion+and+metastasis.">The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis.</a> Nat. Cell Biol. 10 (2), 202-210 (2008).</li><li>Lichner, Z., Pall, E., Kerekes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+miR-290-295+cluster+promotes+pluripotency+maintenance+by+regulating+cell+cycle+phase+distribution+in+mouse+embryonic+stem+cells.">The miR-290-295 cluster promotes pluripotency maintenance by regulating cell cycle phase distribution in mouse embryonic stem cells.</a> Differentiation. 81 (1), 11-24 (2011).</li><li>Subramanyam, D., Lamouille, S., Judson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+targets+of+miR-302+and+miR-372+promote+reprogramming+of+human+fibroblasts+to+induced+pluripotent+stem+cells.">Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells.</a> Nat. Biotechnol. 29 (5), 443-448 (2001).</li><li>Lim, L. P., Lau, N. C., Garrett-Engele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+shows+that+some+microRNAs+downregulate+large+numbers+of+target+mRNAs.">Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.</a> Nature. 433 (7027), 769-773 (2005).</li><li>Huntzinger, E., Izaurralde, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+by+microRNAs:+contributions+of+translational+repression+and+mRNA+decay.">Gene silencing by microRNAs: contributions of translational repression and mRNA decay.</a> Nat. Rev. Genet. 12 (2), 99-110 (2011).</li><li>Thomas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desperately+seeking+microRNA+targets.">Desperately seeking microRNA targets.</a> Nat. Struc. Mol. Biol. 17 (10), 1169-1174 (2010).</li><li>Baek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+microRNAs+on+protein+output.">The impact of microRNAs on protein output.</a> Nature. 455 (7209), 71-71 (2008).</li><li>Guo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+microRNAs+predominantly+act+to+decrease+target+mRNA+levels.">Mammalian microRNAs predominantly act to decrease target mRNA levels.</a> Nature. 466 (3708), 835-840 (2010).</li><li>Ma, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+invasion+and+metastasis+initiated+my+microRNA-10b+in+breast+cancer.">Tumour invasion and metastasis initiated my microRNA-10b in breast cancer.</a> Nature. 449 (7163), 682-688 (2007).</li><li>Visone, R., 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=MicroRNAs+miR-221+and+miR-222,+both+overexpressed+in+human+thyroid+papillary+carcinomas,+regulate+p27Kip1+protein+levels+and+cell+cycle.">MicroRNAs miR-221 and miR-222, both overexpressed in human thyroid papillary carcinomas, regulate p27Kip1 protein levels and cell cycle.</a> Endocrine-Related Cancer. 14 (3), 791-798 (2007).</li><li>Lu, Z., 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=MicroRNA-21+promotes+cell+transformation+by+targeting+the+programmed+cell+death+4+gene.">MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene.</a> Oncogene. 27 (31), 4373-4379 (2008).</li><li>Guo, 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=The+noncoding+RNA,+miR-126,+suppresses+the+growth+of+neoplastic+cells+by+targeting+phosphatidylinositol+3-kinase+signaling+and+is+frequently+lost+in+colon+cancers.">The noncoding RNA, miR-126, suppresses the growth of neoplastic cells by targeting phosphatidylinositol 3-kinase signaling and is frequently lost in colon cancers.</a> Genes Chrom. Cancer. 47 (11), 939-946 (2008).</li><li>Yamakuchi, 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=miR-34a+repression+of+SIRT1+regulates+apoptosis.">miR-34a repression of SIRT1 regulates apoptosis.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (36), 13421-13426 (2008).</li><li>Wickramasinghe, N. 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=Estradiol+downregulates+miR-21+expression+and+increases+miR-21+target+gene+expression+in+MCF-7+breast+cancer+cells.">Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells.</a> Nucleic Acid Res. 37 (8), 2584-2595 .</li><li>Ciaudo, 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=Highly+dynamic+and+sex-specific+expression+of+microRNAs+during+early+ES+cell+differentiation.">Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation.</a> PLoS Genetics. 5, e1000620 (2009).</li><li>Vallejo, D. 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=Targeting+Notch+signaling+by+the+conserved+miR-8/200+microRNA+family+in+development+and+cancer+cells.">Targeting Notch signaling by the conserved miR-8/200 microRNA family in development and cancer cells.</a> EMBO J. 30 (4), 756-769 (2011).</li><li>Jafarifar, F., 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=Repression+of+VEGFA+by+CA-rich+element-binding+microRNAs+is+modulated+by+hnRNAP.">Repression of VEGFA by CA-rich element-binding microRNAs is modulated by hnRNAP.</a> L. EMBO J. 30 (7), 1324-1334 (2011).</li><li>Clancy, J. 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=mRNA+isoform+diversity+can+obscure+detection+of+miRNA-mediated+control+of+translation.">mRNA isoform diversity can obscure detection of miRNA-mediated control of translation.</a> RNA. 17 (6), 1025-1033 (2011).</li></ol>

الكتاب تعلن العلاقات المالية مع المؤسسات التجارية التي لديها مصلحة في العمل المقدم.

microRNAs هي 20-24 RNAs والنوكليوتيدات التي تنظم التعبير الجيني في مرحلة ما بعد transcriptionally التي تحول دون ترجمة مرنا، وبشكل متكرر، زعزعة استقرار مرنا المستهدفة (النظر في [6]). ويجوز للميرنا واحدة تنظم عدة مئات من mRNAs للسيطرة على استجابة الخلية لمؤشرات التنموية والبيئية. تحديد والتحقق من صحة mRNAs هدف أساسي في تحديد دور ميرنا وظيفة في هذه المسارات. ومع ذلك، وتحديد الهدف ليست مباشرة، لأنه في الحيوانات، miRNAs والمواقع المستهدفة ليست متكاملة تماما. في &quot;البذور&quot; المنطقة، والقواعد من 2 إلى 7 من نهاية 5&#39;-من ميرنا، عادة ما تكون مكملة لأهدافها. ومع ذلك، هناك العديد من الاستثناءات لقاعدة البذور، والمصب قاعدة الاقتران يمكن أن تعوض عن مباراة البذور ناقص. وقد تم وضع عدد من خوارزميات الكمبيوتر للتنبؤ اهداف ميرنا على أساس مطابقة البذور والتعويض المصب، والبنية الهدف 1د موقف، والحفاظ على تسلسل، وغيرها من المعالم المختلفة التي لوحظت للأهداف التحقق تجريبيا (مراجعة في [7]). بينما في silico التنبؤات هي مريحة وتفعل الكثير من تحديد أهداف ميرنا ساري المفعول، وتوقع معظم الجينات فشل التحقق من صحة الاختبارات التجريبية، وعدم توقع الكثير من الأهداف الفعلية. وعلاوة على ذلك، منذ وتستند خوارزميات الكمبيوتر في ملامح الهدف التي سبق تحديدها، أنها لا تسمح باكتشاف الأهداف التي تحيد عن ما هو معروف بالفعل. وقد تم استخدام عدد من النظم التجريبية بنجاح لتحديد أو اكتشاف الأهداف ميرنا وظيفية في الخلايا الحية. هذه الأساليب تشمل الفحص العالمية ميكروأرس الحمض النووي الريبي والتسلسل، RNA المشترك مناعي (RIP)، ومستقرة وصفها النظائر مع الأحماض الأمينية في ثقافة خلية (SILAC)، وهي طريقة البروتين (مراجعة في [7]). كل أسلوب له مزاياه وعيوبه. منذ ميرنا تستهدف زعزعة الاستقرار في كثير من الاحيان مرنا ويؤدي الى تدهورها، يا خسارةويمكن البحث في زيادة مستوى مرنا بعد إدخال ميرنا تقليد أو المانع، على التوالي، وتحديد الأهداف ميرنا. تم الكشف بسهولة هذه الخسارة أو الربح من مرنا من قبل ميكروأري أو تسلسل عميق. في حين طرق الكشف مرنا هي أبسط وأكثر حساسية من طرق الكشف البروتين، والكشف عن مرنا يغيب عن أي أهداف ميرنا التي ليست متدهورة. التقارير الأخيرة من مختبر Bartell مقارنة النتائج المستهدفة من الكشف عن الحمض النووي الريبي ميرنا مع هؤلاء من SILAC [8] أو التنميط الريبوسوم [9] إشارة إلى أن miRNAs الثدييات التصرف في الغالب عن طريق خفض مستويات مرنا الهدف. ومع ذلك، فقد اكتشفت مختبرات أخرى كثيرة تعمل مع الأهداف الفردية ميرنا تغير في البروتين ولكن أي تغيير في مستوى مرنا. تقارير ما يبدو أن تنظيم متعدية مع عدم فقدان مرنا للكشف ما يلي: مير 10B في HOXD10 [10]، miR221/222 على p27kip1 [11]، مير-21 على Pdcd4 [12]، مير 126 في p85β [13] مير-34 على SIRT1 [14]، مير-21 على PTEN [15]، مير 302d في Arid4b [16]، مير 200C على JAG1 [17]، 567 مير 299، 297، و، والثانية 609 في VEGFA [18]. وعلاوة على ذلك، كلانسي وآخرون [19] ذكرت مؤخرا ان تم الكشف عن تنظيم متعدية فقط بواسطة Let-7 عندما تم الكشف عن الأشكال الإسوية مرنا الفردية التي تحتوي على موقع دعونا-7 الهدف بشكل انتقائي. هذه الأخيرة تشير إلى أنه، على الأقل في بعض الحالات، يمكن التغاضي عن تنظيم متعدية في لمحة مركب - وهذا هو، عندما يتم الكشف عن جميع الأشكال الإسوية مرنا واحدة مرنا - التي تم الحصول عليها كما هو الحال مع العديد من ميكروأري والتحليلات تسلسل. وسوف يشارك في مناعي من ميرنا، مرنا المجمعات عبر argonaute (ago2 عادة) أو بروتين آخر يرتبط (الحمض النووي الريبي مناعي، أو RIP) عزل أهداف ميرنا بغض النظر عن آلية التنظيم. وبالإضافة إلى ذلك، RIP بالكشف عن الذاتية، ويفترض من الناحية البيولوجية ذات الصلة، وتفاعلات. ومع ذلك، وليس من المعروف ما إذا كانت جميع التفاعلات ميرنا، مرنا وظيفية، وRIP من شأنه أن يفوت أي أهداف مرنا أن أضم عابر فقط أو تتدهور بسرعة. وعلاوة على ذلك، لا بد من استنتاج محدد ميرنا-مرنا شركاء bioinformatiلأن ساتيا جميع أزواج ميرنا-مرنا شارك في عجل معا. أخيرا، SILAC يحدد مباشرة على المنتج النهائي للتنظيم ميرنا، وهو البروتين نفسه، ولكن غير حساس ويفتقد بالتالي البروتينات النادرة والتغييرات أضعاف صغيرة في مستويات البروتين. في ضوء القيود الحالية مع الأساليب ميرنا تحديد الهدف، وشعرنا بان من الضروري ايجاد فحص إضافي العالمي لتحديد أهداف ميرنا الفنية من خلال آلية بديلة. لتلبية هذه الحاجة، مرخصة لدينا التكنولوجيا التي اخترعها Gaken يوب والعظيم Mohamedali من كينجز كوليدج بلندن. اختراعهم هو التحام اختيار مزدوج البروتين، وعلى وجه التحديد، وكيناز-zeocin ثيميدين الانصهار، والتي تنظمها مكتبة [كدنا] من احتمال تسلسل الهدف ميرنا في 3&#39;UTR لها. ويمكن تحديد خلايا transfected ثابت مع والتعبير عن TKzeo، [كدنا] يبني مع zeocin، كما هو موضح في الشكل رقم 2. بعد تعبر عن ميرنا من مصلحة، يمكن تحديد الأهداف التي ميرنا ووايganciclovir عشر، كما هو موضح في الشكل رقم 3. Ganciclovir يقتل الخلايا معربا عن كيناز ثيميدين، وهذا هو، أي الخلايا معربا عن TKzeo، [كدنا] تفتقر هدفا لميرنا من الفائدة. ويمكن أن تكون معزولة استهدفت [كدنا] من التضخيم PCR من الحمض النووي من الخلايا التي البقاء على قيد الحياة باستخدام ganciclovir الاشعال التي يمكن أن تكون متسلسلة الجناح المنتجات [كدنا]، وPCR لتحديد الأهداف. لقد قمنا بتطوير تكنولوجيا كلية الملك إلى وسيلة جديدة لتحديد الهوية العالمية، واكتشاف أهداف وظيفية ميرنا الإنسان - معرف الهدف المكتبة البعثة. مع الهدف المكتبة الهوية، ويمكن للمستخدمين عزل أهداف ميرنا من خلال سلسلة من الثدييات خلية ترنسفكأيشن الثقافة وخطوات اختيار الدواء. مكتبة شاملة، ويحتوي على 66-79٪ من الجينات البشرية. النتائج الأولية تشير إلى أن كلا اكتشف سابقا، وكذلك يمكن أن تكون معزولة أهداف جديدة من معرف المكتبة بعثة الهدف. على الرغم من أن البروتوكول هو من بعض طول،استخدم هدف المكتبة رقم قياسي يتطلب تقنيات مختبر الجزيئية - زراعة الخلايا الثديية، ترنسفكأيشن، واختيار الدواء، PCR، والتسلسل - وبالتالي، ينبغي أن يكون جيدا في وسائل معظم علماء الأحياء. نقترح ان يتم إيلاء الاهتمام الكافي لتصميم التجارب المناسبة، وتحسين ظروف والثقافة، والأهم من ذلك، قبل اختبار كل نوع من الخلايا الجديدة لتحديد مستويات الدواء الأمثل للحصول على خطوات اختيار (قتل، منحنيات) لضمان نجاح مع الهدف المكتبة الهوية. كما هو الحال مع جميع وسائل ميرنا تحديد الهدف، ومن المستحسن جدا أن يتم التحقق من صحة الأهداف التي تم تحديدها عن طريق فحص مكتبة معرف الهدف مع الأسلوب الثاني، مثل [ميكروأري، qRT PCR، مراسل فحص (أي luciferase المراسل)، أو تحليل الغربية.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> في Jumpstart REDTaq للخرسانة الجاهزة </td><td> سيغما الدريخ </td><td> P0982 </td><td></td></tr><tr><td> Ganciclovir </td><td> سيغما الدريخ </td><td> G2536 </td><td></td></tr><tr><td> G418 </td><td> سيغما الدريخ </td><td> G8168 </td><td></td></tr><tr><td> بوروميسين </td><td> سيغما الدريخ </td><td> P9620 </td><td></td></tr><tr><td> توبو TA </td><td> إينفيتروجن </td><td> KNM4500-01 </td><td></td></tr><tr><td> GenElute الثدييات الجينوم الحمض النووي Miniprep كيت </td><td> سيغما الدريخ </td><td> G1N10 </td><td></td></tr><tr><td> ك خلية Nucleofection محددةهذا </td><td> Lonza </td><td></td><td></td></tr><tr><td> Nucleofector </td><td> Lonza </td><td> AAD-1001 </td><td></td></tr><tr><td> Zeocin </td><td> إينفيتروجن </td><td> <a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=R25001">R250-01</a> </td><td></td></tr><tr><td height="1"> الهدف رقم المكتبة </td><td> سيغما الدريخ </td><td> MREH01 </td><td></td></tr></tbody></table>

genome-wide screen for mirna targets using the mission target id library

تم تصميم المكتبة الرقم المستهدف للمساعدة في اكتشاف وتحديد الأهداف من microRNA (ميرنا). مكتبة معرف الهدف هو البلازميد ومقرها الجينوم على نطاق مكتبة [كدنا] المستنسخة في اتجاه مجرى النهر 3&#39;UTR من انصهار بروتين ثنائي اختيار، ثيميدين كيناز-zeocin (TKzeo). الجولة الأولى من اختيار هو لtransformants مستقرة، تليها مع الأخذ ميرنا من الفائدة، وأخيرا، واختيار لcDNAs تحتوي على هدف وميرنا و. ويتم تحديد cDNAs مختارة من قبل التسلسل (انظر الشكل 1-3 لمعرف الهدف سير العمل المكتبات والمعلومات). لضمان تغطية واسعة من transcriptome الإنسان، تم إنشاؤها الهدف رقم مكتبة cDNAs عبر بنسبة ضئيلة من DT فتيلة باستخدام مجموعة من الحمض النووي الريبي مجموع المحضرة من أنسجة بشرية متعددة وخطوط الخلية. مما أدى مجموعة [كدنا] 0،5-4 كيلو بايت، مع متوسط ​​حجم 1.2 كيلوبايت، وكانت مستنسخة في البلازميد المزدوج اختيار p3TKzeo (انظر الشكل 4 لخريطة البلازميد). الأهداف الجينات ممثلة في لىويمكن الاطلاع على الموقع الشبكي لbrary سيغما الدريخ. نتائج من التسلسل البورشيد (الجدول 3)، وتبين أن المكتبة تضم 16922 من 21518 جينات فريدة من نوعها في RefGene UCSC (79٪)، أو 14000 الجينات مع 10 أو أكثر يقرأ (66٪).

Watch this Scientific Journal Video about Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library at JoVE.com

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

مكتبة معرف الهدف هو البلازميد ومقرها الجينوم على نطاق جمع من [كدنا] المستنسخة يستخدم لتحديد أهداف ميرنا. هنا علينا أن نبرهن كيفية استخدامه وتطبيقه.

الجينوم على نطاق الشاشة لأهداف ميرنا باستخدام معرف الهدف المكتبة MISSION

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

genome-wide-screen-for-mirna-targets-using-mission-target-id

Nanyang Technological University

Research

JoVE Journal

Biology

17.5K Views.  Sigma-Aldrich. مكتبة معرف الهدف هو البلازميد ومقرها الجينوم على نطاق جمع من [كدنا] المستنسخة يستخدم لتحديد أهداف ميرنا. هنا علينا أن نبرهن كيفية استخدامه وتطبيقه.

Video: Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

الجينوم على نطاق تحليل المستويات (شحن) من الحمض الريبي النووي النقال باستخدام Aminoacylation ميكروأرس]

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

الجينوم على نطاق التحليل باستخدام رقاقة لتحديد أهداف محددة الجينات شكل الإسوي

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

LentiPlex بعثة مجمع shRNA الفرز مكتبة في خلايا الثدييات

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

الكمية والآلي عالية الإنتاجية الجينوم على نطاق شاشات رني في C. ايليجانس</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of protein-bound DNA and aligning the short reads to a reference genome. Enriched regions are revealed as peaks, which often differ dramatically in shape, depending on the target protein1. For example, transcription factors often bind in a site- and sequence-specific manner and tend to produce punctate peaks, while histone modifications are more pervasive and are characterized by broad, diffuse islands of enrichment2. Reliably identifying these regions was the focus of our work.
Algorithms for analyzing ChIPseq data have employed various methodologies, from heuristics3-5 to more rigorous statistical models, e.g. Hidden Markov Models (HMMs)6-8. We sought a solution that minimized the necessity for difficult-to-define, ad hoc parameters that often compromise resolution and lessen the intuitive usability of the tool. With respect to HMM-based methods, we aimed to curtail parameter estimation procedures and simple, finite state classifications that are often utilized.
Additionally, conventional ChIPseq data analysis involves categorization of the expected read density profiles as either punctate or diffuse followed by subsequent application of the appropriate tool. We further aimed to replace the need for these two distinct models with a single, more versatile model, which can capably address the entire spectrum of data types.
To meet these objectives, we first constructed a statistical framework that naturally modeled ChIPseq data structures using a cutting edge advance in HMMs9, which utilizes only explicit formulas-an innovation crucial to its performance advantages. More sophisticated then heuristic models, our HMM accommodates infinite hidden states through a Bayesian model. We applied it to identifying reasonable change points in read density, which further define segments of enrichment. Our analysis revealed how our Bayesian Change Point (BCP) algorithm had a reduced computational complexity-evidenced by an abridged run time and memory footprint. The BCP algorithm was successfully applied to both punctate peak and diffuse island identification with robust accuracy and limited user-defined parameters. This illustrated both its versatility and ease of use. Consequently, we believe it can be implemented readily across broad ranges of data types and end users in a manner that is easily compared and contrasted, making it a great tool for ChIPseq data analysis that can aid in collaboration and corroboration between research groups. Here, we demonstrate the application of BCP to existing transcription factor10,11 and epigenetic data12 to illustrate its usefulness.

Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment.

رواية النظرية الافتراضية تغيير نقطة حسابي لتحليل الجينوم-اسعة من أنواع البيانات المتنوعة ChIPseq

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

حذوفات الجينات في الجينوم على نطاق<em&gt; العقدية الدم</em&gt; من جانب PCR إنتاجية عالية

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

Polysome تجزئة وتحليل الثدييات Translatomes على مقياس الجينوم على نطاق

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

الكشف عن أهداف ميرنا في إنتاجية عالية باستخدام الفحص 3&#39;LIFE

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

على صعيد رسم الخرائط الجينوم من المخدرات-DNA التفاعلات في الخلايا مع COSMIC (يشابك من الجزيئات الصغيرة لعزل لونين)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

تنقية الجينوم على نطاق خارج الصبغي التعميم الحمض النووي من خلايا حقيقية النواة

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...