المهمة هي علامة تجارية مسجلة لشركة التكنولوجيا الحيوية سيغما الدريخ LentiPlex هي علامة تجارية لشركة التكنولوجيا الحيوية سيغما الدريخ

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	<li>Rao, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+vs.+shRNA:+similarities+and+differences.">siRNA vs. shRNA: similarities and differences.</a> Adv. Drug Deliv. Rev. 61 (9), 746-746 (2009).</li><li>Root, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+loss-of-function+screening+with+a+lentiviral+RNAi+library.">Genome-scale loss-of-function screening with a lentiviral RNAi library.</a> Nat Methods. 3 (9), 715-715 (2006).</li><li>Fan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+antitumor+activity+of+TRAIL+combined+with+chemotherapeutic+agents+in+A549+cell+lines+in+vitro+and+in+vivo.">Synergistic antitumor activity of TRAIL combined with chemotherapeutic agents in A549 cell lines in vitro and in vivo.</a> Cancer Chemother Pharmacol. 55, 189-189 (2005).</li><li>Ji, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+screen+of+shRNAs+targeting+tumor+suppressor+genes+to+identify+factors+involved+in+A549+paclitaxel+sensitivity.">A screen of shRNAs targeting tumor suppressor genes to identify factors involved in A549 paclitaxel sensitivity.</a> Oncology Reports. 18, 1499-1499 (2007).</li><li>Fillmore, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+expands+breast+cancer+stem-like+cells+through+paracrine+FGF/Tbx3+signaling.">Estrogen expands breast cancer stem-like cells through paracrine FGF/Tbx3 signaling.</a> Proc. Natl. Acad. Sci. U.S.A. 14 (50), 21737-21737 (2010).</li><li>Bhardwaj, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+protein+phosphatase+2A+activity+alters+androgen-independent+growth+of+prostate+cancer+cells:+therapeutic+implications.">Modulation of protein phosphatase 2A activity alters androgen-independent growth of prostate cancer cells: therapeutic implications.</a> Mol. Cancer. Ther. 10 (5), 720-731 (2011).</li><li>Cheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AKT2,+a+putative+oncogene+encoding+a+member+of+a+subfamily+of+protein-serine/threonine+kinases,+is+amplified+in+human+ovarian+carcinomas.">AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas.</a> Proc. Natl. Acad. Sci. U.S.A. 89 (19), 9267-9267 (1992).</li><li>Fernandez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt1+and+Akt2:+differentiating+the+aktion.">Akt1 and Akt2: differentiating the aktion.</a> Histol. Histopathol. 26 (5), 651-651 (2011).</li><li>Luo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+RNAi+screen+identifies+multiple+synthetic+lethal+interactions+with+the+Ras+oncogene.">A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene.</a> Cell. 137 (5), 835-835 (2009).</li><li>Ketela, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+platform+for+highly+multiplexed+mammalian+functional+genetic+screens.">A comprehensive platform for highly multiplexed mammalian functional genetic screens.</a> B.M.C. Genomics. 12 (1), 213-213 (2011).</li></ol>

ويعمل ماثيو جيه Coussens ، كورمان كورتني واشلي فيشر L. ، ساجو جاك ، وجون Swarthout بواسطة سيغما الدريخ ، مما يجعل من الأدوات والكواشف المستخدمة في هذه المقالة.

في هذه الدراسة ، فإننا نقدم شاشة الجينوم واسعة لتحديد الجينات المسؤولة عن السمسة A549 من الخلايا التالية باكليتاكسيل / تريل العلاج. استخدام نهج المجمعة في شاشة الجينوم على نطاق يقلل من الوقت والتكلفة والاستثمار في المعدات التي ترتبط عادة مع الشاشات التقليدية التي تم حشدها. حاسمة بالنسبة لنجاح التجارب الشاشة هي الأمثل يؤديها سلفا. يجب أن تكون الظروف تنبيغ وزارة الداخلية تحدد تجريبيا. وينبغي اختيار الأسلوب يسمح للاختيار من الخلايا القوية التي تعرض النمط الظاهري المطلوب (على سبيل المثال ، والبقاء التعبير سطح البروتين ، الخ). والاستفادة المثلى من هذه المعلمات تقليل عدد ايجابيات كاذبة الكشف عنها. هنا ، كان حصادها gDNA من سكان الجزء الأكبر من الخلايا المحددة ثم TOPO المستنسخة لتحديد إدراج shRNA الفردية. كان من الممكن تحسين واحدة لهذه التجربة لتحديد الخلايا الحية لاستخدام نظام مراقبة الأصول الميدانية لضمانيتم جمع إلا أن الخلايا الحية. وسيتم التحقق من الأهداف التي حددها تنبيغ مع shRNAs محددة الهدف في مقايسة الفرز. ويضرب عرض النمط الظاهري صحيحا في معظم الآبار. مكتبة LentiPlex مجمع يتسم بالمرونة في تطبيقها. وهو متوافق مع مجموعة واسعة من التطبيقات المصب ، ويمكن استخدامها مع استراتيجيات الاختيار إما إيجابية أو سلبية. تبعا للظروف الفرز ، ويتم إنجاز deconvolution لإدراج shRNA عبر PCR / الاستنساخ ، ميكروأري ، أو الجيل القادم التسلسل 9 و 10 وبينما هو راسخ في قوة وسرعة من هذه التقنيات لdeconvolute شاشات رني ، واحدة مع القلق وميكروأري الجيل القادم من أساليب التسلسل هو توافر الموارد المعلوماتية الحيوية والمعارف اللازمة لتحليل وتفسير النتائج بشكل صحيح التجريبية. يمكن أن تكون تكاليف هذه التقنيات المرتبطة في كثير من الأحيان يكون عائقا أمام القيام شاشات الجينوم واسعة. APCR / استنساخ النهج القائم ، في حين انها لم تكن بقوة ميكروأرس وتسلسل الجيل المقبل من هو بديل فعال وبتكلفة أقل. اجيلنت يقدم الآن مجموعة والمخصص اعتمادا على مكتبة shRNA TRC لتقديم المزيد من المساعدات في شاشات LentiPlex. هذه الخيارات تسمح للباحثين استخدام LentiPlex لدراسة مجموعة متنوعة من الوظائف الخلوية.

<html><table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الكاشف </td><td> شركة </td><td> كتالوج رقم </td></tr><tr><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=SHPH01%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">صفيف LentiPlex المجمعة</a> </td><td> <a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">سيغما الدريخ</a> </td><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=SHPH01%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">SHPH01</a> </td></tr><tr><td> خلايا A549 </td><td> ATCC </td><td> CCL - 185 </td></tr><tr><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=T5694%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">TRAIL</a> </td><td> <a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">سيغما الدريخ</a> </td><td><html> oductDetail.do؟ lang = ar في T5694 و N4 = ٪ 7CSIGMA وN5 = SEARCH_CONCAT_PNO ٪ 7CBRAND_KEY &amp; F = SPEC &quot;&gt; T5694 </td></tr><tr><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=T7402%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">باكليتاكسيل</a> </td><td> <a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">سيغما الدريخ</a> </td><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=T7402%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">T7402</a> </td></tr><tr><td> توبو TA الاستنساخ كيت </td><td> Invitrogen </td><td> K4575 - 01 </td></tr><tr><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=P2893%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">Jumpstart من طق للخرسانة الجاهزة</a> </td><td> <a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">سيغما الدريخ</a> </td><td>ONCAT_PNO 7CBRAND_KEY ٪ &amp; F = SPEC &quot;P2893&gt; </td></tr><tr><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=PLN70%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">GenElute عدة Miniprep البلازميد</a> </td><td> <a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">سيغما الدريخ</a> </td><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=PLN70%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">PLN70</a> </td></tr><tr><td> EcoR1 </td><td> نيو انغلاند Biolabs </td><td> R0101 </td></tr><tr><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=H9268%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">hexadimethrine بروميد</a> </td><td> <a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">سيغما الدريخ</a> </td><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=H9268%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">H9268</a> &lt;/ TD&gt; </tr></tbody></table>

mission lentiplex pooled shrna library screening in mammalian cells

تدخل الحمض النووي الريبي (رني) هو آلية جوهرية الخلوية لتنظيم التعبير الجيني. تسخير قوة فطرية لهذا النظام يتيح لنا ضربة قاضية لمستويات التعبير الجيني في فقدان دراسات وظائف الجينات. هناك طريقتين رئيسيتين لأداء رني. الأولى هي استخدام الصغيرة RNAs والتدخل (siRNAs) التي تم توليفها كيميائيا ، والثاني يستخدم قصيرة القاسي الرناوات (shRNAs) المشفرة داخل البلازميدات 1. يمكن transfected الأخير في الخلايا مباشرة أو حزم جسيمات في تكرار lentiviral غير كفء. المزايا الرئيسية لاستخدام shRNAs lentiviral هو سهولة إدخالها في مجموعة واسعة من أنواع الخلايا ، وقدرتهم على الاندماج في جينوم ثابت لفترة طويلة الأجل واختيار الجينات ضربة قاضية ، ومدى فعاليتها في إجراء عالية الإنتاجية من الشاشات فقدان الوظيفة. لتسهيل هذا وضعنا LentiPlex المجمعة shRNA المكتبة. وMISسيون مكتبة LentiPlex shRNA الإنسان مجمع بركة الجينوم على نطاق lentiviral المنتجة باستخدام عملية الملكية. مكتبة يتكون من بنيات shRNA أكثر من 75،000 من الهيئة تستهدف جمع 15000 + 2 الجينات البشرية. يتم اختبار كل مكتبة للتمثيل shRNA قبل الافراج المنتج لضمان تغطية قوية المكتبة. وتقدم المكتبة في شكل جاهز للاستخدام في lentiviral التتر ما لا يقل عن 5 × 10 8 مل / TU عبر فحص P24 وقبل مقسمة إلى عشرة subpools حوالي 8000 shRNA يبني كل منهما. كما يتم توفير التضخيم وتسلسل الاشعال لتحديد الهدف المصب. أثبتت الدراسات السابقة نشاط انتيتومور المتآزر لTRAIL عندما يقترن باكليتاكسيل A549 في الخلايا ، وسرطان الرئة الخلية البشرية السطر 3 ، 4. في هذه الدراسة تثبت أننا في تطبيق مكتبة LentiPlex shRNA المجمعة لإجراء سريعا شاشة اختيار إيجابية للجينات تشارك في السمسة سو A549 الخلايا عند تعرضها للتريل وباكليتاكسيل. حاجز واحد واجه كثير من الأحيان مع شاشات عالية الإنتاجية والتكلفة وصعوبة في deconvolution ، ونحن من التفصيل أيضا نهجا فعالا من حيث التكلفة النسائل المتعددة باستخدام التسلسل التقليدي.

Watch this Scientific Journal Video about MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells at JoVE.com

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

هنا نستخدم مكتبة LentiPlex الإنسان المجمعة التقليدية وأساليب التسلسل الجيني لتحديد أهداف تعزيز بقاء الخلية. نحن لشرح كيفية إعداد وdeconvolute شاشة LentiPlex والتحقق من صحة النتائج.

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 ...

mission-lentiplex-pooled-shrna-library-screening-in-mammalian-cells

Research

JoVE Journal

Biology

18.5K Views.  Sigma-Aldrich. هنا نستخدم مكتبة LentiPlex الإنسان المجمعة التقليدية وأساليب التسلسل الجيني لتحديد أهداف تعزيز بقاء الخلية. نحن لشرح كيفية إعداد وdeconvolute شاشة LentiPlex والتحقق من صحة النتائج.

Video: MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing Subculturing وخلايا الثدييات

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

esiRNA بعثة لفحص رني في خلايا الثدييات

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

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

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 cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

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, ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

تحليل المركز دورة الخلية في خلايا الثدييات

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

تصور mRNAs والشبكة الإندوبلازمية في خلايا الثدييات المترجمة

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

سيرنا فحص لتحديد اليوبيكويتين واليوبيكويتين مثل المنظمين نظام مقررات تمهيدية البيولوجية في خلايا الثدييات مثقف

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

قياس مستويات هيم في تخليق خلايا الثدييات

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

فحوصات مراسل المتوازية نطاق واسع في خلايا الثدييات مثقف

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

التصوير المحلية الكالسيوم<sup&gt; 2+</sup&gt; الإشارات في خلايا الثدييات مثقف

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

توجه طريقة تطور في<em&gt; خميرة الخباز</em&gt;: المسخ مكتبة الخلق والفرز

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...