Name: true gene silencing: screening of a heptamer-type small guide rna library for potential cancer therapeutic agents
Uploaded: 2016-06-02T14:00:00.000+00:00
Duration: 9 min 18 s
Description: Watch this Scientific Journal Video about TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents at JoVE.com

Bu çalışma Hedef odaklı Ar-Ge, Japonya Bilim ve Teknoloji Ajansı, Japonya Özel Okullar Tanıtım ve Yardımlaşma Corporation Bilim Araştırma Teşvik Fonu aracılığıyla Uyarlanabilir ve Dikişsiz Teknoloji Transferi Programı tarafından desteklenen ve JSPS KAKENHI Hibe Numaraları 24300342 ve 15H04313 oldu .

Bir heptamer tipi 1. İnşaat sgRNA Kütüphanesi <ol><li> Tam ölçekli bir kütüphane inşa edilecek sürece 16.384 (4 7) 7-nt dizilerinin bir alt kümesini seçin. Not: dizileri rasgele ve / veya belirli bir hücre RNA&#39;lar hedeflemek için tasarlanmış olabilir. İkincisi, saç tokası yapıları 4,10 hemen alt baş 7-nt dizilere tamamlayıcı uygun bir bilgisayar programı ve / veya görsel olarak basıp dizilerin yardımı ile, bir hedef RNA tRNA T kolunu benzer saç tokası yapıları bulmak 17,18. </li><li> Seçilen heptamerlerin her sentezleme tam 2&#39;- O metillenmiş, 5&#39;- ve bir DNA / RNA sentezleyici ile 3&#39;-fosforile edilmiş RNA. Kontrollü gözenekli cam (CPG) Zemin üzerinde fosforamidit yöntemini kullanarak, ve 5&#39;-terminal 4,4&#39;-dimetoksitritil (DMT) sentezin son döngüde (5 &#39;fosfat ilave) koruma grubu terk 23. NOT: Özel synthetic RNA da oligonükleotid tedarikçilerden elde edilebilir. </li><li> Son sentez döngüsünün tamamlanmasından sonra, 8 saat boyunca 55 ° C &#39;de, 1 ml 29%&#39; si amonyum hidroksit ekleyin Teflon astar vidalı kapaklı bir cam şişeye sütun sentezlenen oligonükleotid ile CPG transferi ve inkübe CPG, oligonükleotidin ayrılması ve bazlar ve fosfatlar koruma gruplarını kaldırmak. </li><li> 40 ° C &#39;de santrifüj lü bir evaporatörde amonyum hidroksit buharlaştınn ve 0.2 ml 0.1 M n-hexylammonium asetat içinde oligonükleotid yeniden çözülür. </li><li> % 10-50 asetonitril / 0.1 M n-hexylammonium asetat ihtiva eden bir tampon maddesi ile bir polistiren-divinilbenzen kolonu kullanılarak ters-faz yüksek performans sıvı kromatografi yoluyla sentetik oligonükleotit saflaştınlır. 80 ° C de 25 dakika süreyle / 2.0 ml dk sütun çalıştırın. </li><li> % 20 asetik asit solüsyonu yapmak için kısımlara ayrılmış örnek (1-2 mi) konsantre asetik asit 0.25-0.5 ml ilave edilir, ve doğmuş iyileşmesinite oda sıcaklığında 1 saat boyunca bu çözelti, DMT grubu çıkarmak için. </li><li> 40 ° C &#39;de santrifüj lü bir evaporatörde örnek kurutun, 1 mi 29% amonyak içinde yeniden çözülür ve 5&#39; fosfat yan zincirini çıkarmak için oda sıcaklığında 15 dakika boyunca çözeltiden inkübe edin. </li><li> 40 ° C &#39;de santrifüj lü bir evaporatörde 0.3 ml numune hacmi (1 mi) azaltır. </li><li> kesme 1000, 4 ° C&#39;de bir gece boyunca moleküler ağırlığa sahip bir diyaliz tüpü kullanılarak damıtılmış su karşı tüm konsantre edildi örneği (500 mi) diyaliz. </li><li> Daha sonra, bir pipet ile 1.5 ml tüp diyaliz tüpünden diyaliz örnek aktarın 40 ° C &#39;de santrifüj lü bir evaporatörde kurutun ve hacmi arasında olmalıdır, su için enjeksiyon dereceli su içinde yeniden çözülür daha büyük bir 100 uM çözelti yapmak için yeteri kadar küçük olabilir. </li><li> bir spektrofotometre ile 260 nm&#39;de RNA numunesinin optik yoğunluğunun ölçülmesi ve 100 olan konsantrasyonunu ayarlamakSu için enjeksiyon dereceli su ilave edilerek uM. -20 ° C Kullanmadan önce aşağıda RNA çözümü saklayın. </li></ol> Hücre Canlılığına için sgRNA Kütüphanesi 2. Eleme <ol><li> Hazırlama 10 3 hücre / 99 ul / göz (ör HL60 ve RPMI-8226),% 10 fetal sığır serumu ve% 1 penisilin-streptomisin çözeltisi ile takviye edilmiş RPMI-1640 ortamı içinde bir 96-yuvalı tabak üzerinde. Sadece arka plan çıkarma için ortam içeren kuyu hazırlayın. numune kuyuları çevreleyen kuyu boş steril su ekleyin, kenar etkilerini ortadan kaldırmak için. </li><li> her bir suda için enjeksiyon dereceli su içinde çözülmüş heptamer tipi sgRNA (100 uM) 1 ul ekle. üç kez, her sgRNA deneyi. 3 gün boyunca% 5 CO2 nemlendirilmiş inkübatör içinde 37 ° C&#39;de inkübe plakası. </li><li> Her bir oyuğa, bir ticari olarak temin edilebilir WST-8 çözeltisinin uygun bir hacmi ekleyin. 1 ila 4 saat boyunca, aynı inkübatörde 37 ° C&#39;de inkübe plakası. </li><li> absorbans a ölçünBir mikro levha okuyucusu ile 450 nm t. NOT: Bir çok durumda, emme ve canlı hücre sayısı arasındaki doğrusal absorbans değeri 1.0 aşmadıkça muhafaza gibi görünmektedir. </li></ol> Akış Sitometrisi ile Etkili sgRNAlardır 3. Değerlendirilmesi <ol><li> Hazırlama 10 3 hücre /% 10 fetal bovin serumu ve% 1 penisilin-streptomisin çözeltisi ile takviye edilmiş RPMI-1640 ortamı içinde bir 96-yuvalı tabak üzerinde 99 ul / göz (ör HL60). bir sgRNA test edilecek en az 10 kuyu hazırlayın. numune kuyuları çevreleyen kuyu boş steril su ekleyin, kenar etkilerini ortadan kaldırmak için. </li><li> her bir suda için enjeksiyon dereceli su içinde çözülmüş heptamer tipi sgRNA (100 uM) 1 ul ekle. 3 gün boyunca% 5 CO2 nemlendirilmiş inkübatör içinde 37 ° C&#39;de inkübe plakası. </li><li> bir pipet, bir 5-ml&#39;lik bir tüp içine en az 10 kuyu aynı sgRNA ile muamele hücreleri toplamak. 30 ° C&#39;de bir santrifüj içinde 5 dakika boyunca tüp spin0 xg ve medya atın. </li><li> tüpe 1X fosfat tamponlu salin (PBS) 2 ml ilave edilir ve hücreler yeniden askıya. 300 x g&#39;de santrifüj içinde 5 dakika boyunca tüp spin ve PBS atın. tüp 1x anneksin V bağlayıcı tampon 150 ul ekleyin ve hücrelerin yeniden askıya. </li><li> , Fikoeritrin 2 ul (PE) ile konjüge edilmiş anneksin V çözeltisi ve tüp 7-aminoactinomycin D (7AAD) çözeltisi 1 ul ekle iyice karıştırın ve karanlıkta, oda sıcaklığında 15 dakika boyunca tüp inkübe edin. 24 debi sitometresinde örnek analiz edin. </li></ol>

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Niigata University of Pharmacy and Applied Life Sciences has a pending patent application on a subset of the heptamer-type sgRNAs.

In most cases, fully 2&#8242;-O-methylated, 5&#8242;- and 3&#8242;-phosphorylated heptamer-type sgRNAs dissolved in water-for-injection-grade water (in the concentration of 100 &#181;M) appear to be stable at below -20 &#176;C for at least one year, judging from their activity to induce apoptosis in cancer cells. However, their stability would change depending on their sequence, quality, and purity. In some cases, 5&#8242;- or 3&#8242;-phosphate of a part of sgRNA molecules appears to be removed spontaneously during storage, judging from mass spectrometry analysis, although this may not affect their activity seriously.
When a hit sgRNA is obtained by the library screening, its cellular target RNAs can be found by transcriptome analysis through a DNA microarray and/or next-generation sequencing. The data obtained so far, however, suggest that downregulation levels of target RNAs via heptamer-type sgRNAs are usually moderate regardless of the presence or absence of transfection reagents10,12,14,17,18. 
The advantage of heptamer-type sgRNAs targeting BCL2 and WT1 mRNAs is that they can efficiently induce apoptosis in human leukemia cells in spite of weak downregulation of the target mRNAs17,18, suggesting the presence of additional RNA targets and/or additional mechanisms other than TRUE gene silencing. 
Since even the screening of the library containing only 156 heptamer-type sgRNAs can make 20 hits, around 2,000 hits would be expected to be obtained by screening the full-scale library composed of 16,384 heptamers22,25. 
For future applications, sgRNAs other than the heptamer type can be more suitable for TRUE gene silencing11-13,15,16, although heptamer-type sgRNAs are easier, more accurate and cheaper to synthesize than longer sgRNAs. Once the therapeutic sgRNA candidate is obtained, it is easy to proceed to animal model experiments.

(TR Nase Z L sonra adlandırılan - u E fficacious gen susmasının tilizing) DOĞRU gen susturma RNA yönelik gen susturma teknolojileri 1 biridir. Bu teknoloji geliştirdi tRNase Z L (ya da 3 &#39;tRNase), tRNA 3&#39; işleme endoribonuclease özelliklerine dayanarak olmuştur: Bir tanıyarak yapay küçük bir rehber RNA (sgRNA) yönetimindeki herhangi beklenen yerinde herhangi bir hedef RNA yarabilir 9 - hedef RNA ve sgRNA 2 arasında oluşturulmuş önceden tRNA benzeri ya da mikro-ön-tRNA-benzeri bir yapı. uzunluğunda nt genellikle 7-31 olan sgRNA, dört gruba, 5&#39;-yarım-tRNA, heptamer RNA, 14-nt lineer RNA ve kanca RNA kategorize edilir. 15 - Biz hücreler 10 canlı içine çeşitli yapay olarak tasarlanmış sgRNAlardır sunarak DOĞRU gen susturma etkinliğini göstermiştir. Biz de sgRNAlardır hücrelerin wi tarafından alınabilir göstermiştirherhangi bir transfeksiyon reaktifleri thout ve hedef RNA seviyelerini bozulduğunu ve / veya insan kanser hücrelerinin 16 apoptosis doğurabileceği - 18. 21 - DOĞRU gen susturma tRNase Z L ve doğal sgRNAlardır 19 üzerinden hücre içi ve hücreler arası gen düzenleyici ağ sistemi üzerinde çalışması için görünür. Kan kanserleri 22 potansiyel terapötik heptamer tipi sgRNAlardır aramak için 156 heptamer tipi sgRNAlardır içeren bir sgRNA kütüphane inşa ettik. İnsan miyeloma ve lösemi hücrelerinin ömrü üzerindeki etkisi için filtrelenmiş ve 20 tanesi etkin kanser hücre çizgilerinin en az biri apoptosis bulmuşlardır. Ve bunların 4 fare ksenogreft deneylerinde tümör büyüme oranlarını azaltmak için gösterilmiştir. Burada kan kanserlerine karşı potansiyel terapötik ilaçların bir heptamer tipi sgRNA kitaplığının taranması için bir protokol mevcut. protokol nasıl dahilhücre canlılığı ve nasıl daha fazla flow sitometri ile etkili sgRNAlardır değerlendirmek için her sgRNA etkisini değerlendirmek için nasıl sgRNA kütüphanesini oluşturmak için. Bunlar, bu protokolde yer almaz, ancak fare ksenogreft modellerinde sgRNA hücresel hedef RNAlar için analiz ve etkili sgRNAlardır değerlendirilmesi, geleneksel yöntemlerle 17,22 göre gerçekleştirilebilir.

<table><tbody><tr><td>Custom heptamer RNA</td><td>Nippon Bioservice</td><td></td><td></td></tr><tr><td>OligoSep Prep HC Cartridge</td><td>Transgenomic</td><td>99-3860</td><td></td></tr><tr><td>Water-for-injection-grade Water</td><td>Otsuka Pharmaceutical&amp;nbsp;</td><td>7131400A2129</td><td></td></tr><tr><td>RPMI-1640 medium</td><td>Wako</td><td>189-02025</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>SIGMA-ALDRICH</td><td>172012-500ML</td><td></td></tr><tr><td>Penicillin-Streptomycin Mixed Solution</td><td>Nacalai Tesque</td><td>26253-84</td><td></td></tr><tr><td>96 Well Plate</td><td>Greiner Bio-one</td><td>655 180</td><td></td></tr><tr><td>Cell Counting Kit-8</td><td>DOJINDO</td><td>343-07623</td><td>WST-8 solution</td></tr><tr><td>Microplate Reader, Sunrise Thermo RC-R</td><td>TEKAN</td><td>510-82851</td><td></td></tr><tr><td>FACS Round-Bottom Tube</td><td>BD Falcon</td><td>60819-820</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>SIGMA-ALDRICH</td><td>P7059-1L</td><td></td></tr><tr><td>PE Annexin V Binding Buffer</td><td>BD Biosciences</td><td>51-66121E</td><td></td></tr><tr><td>PE Annexin V</td><td>BD Biosciences</td><td>51-65875X</td><td></td></tr><tr><td>7AAD</td><td>SIGMA-ALDRICH</td><td>A9400-1MG</td><td></td></tr><tr><td>Flow Cytometer, FACSCalibur</td><td>BD Biosciences</td><td>342973</td><td></td></tr></tbody></table>

true gene silencing: screening of a heptamer-type small guide rna library for potential cancer therapeutic agents

DOĞRU gen susturma (tR Nase Z L sonra adlandırılan - u, e fficacious gen susturulması tilizing) tRNA 3 &#39;işleme endoribonuclease, tRNase Z L rehberlik edecek bir yapay küçük bir rehber RNA (sgRNA) kullanır teknolojileri susturma RNA yönettiği genin, biri bir hedef RNA tanımak için. sgRNAlardır bir transfeksiyon reaktifleri olmayan hücreler tarafından alınabilir ve hedef RNA seviyelerini bozulduğunu ve / veya insan kanser hücrelerinde apoptosisi teşvik edebilir. İnsan miyeloma ve lösemi hücrelerinin canlılığı üzerindeki etki için 156 heptamer tipi sgRNAlardır ihtiva eden bir sgRNA kitaplığı tarandı ve bunların 20 verimli, kanser hücre çizgilerinin en az biri apoptosis bulmuşlardır. Burada kan kanserlerine karşı potansiyel terapötik ilaçların bir heptamer tipi sgRNA kitaplığının taranması için bir protokol mevcut. protokol hücre canlılığı ve her sgRNA etkisini değerlendirmek nasıl fu için nasıl, sgRNA kütüphane oluşturmak için nasıl dahilrther flow sitometri ile etkili sgRNAlardır değerlendirir. Yaklaşık 2000 hit 16.384 heptamerlerin oluşan tam ölçekli sgRNA kütüphanenin taranması ile elde edilebilir olması beklenmektedir.

Altı heptamer tipi sgRNAlardır 5&#39;-AUCUUCA-3 &#39;(H1885), 5&#39;-ACACACA-3&#39; (H3277), 5&#39;-GGGGGCG-3 &#39;(H10927), 5&#39;-GGGGCCC-3&#39; (H10944), 5&#39;-GCCCCCG-3 &#39;(H12287), ve 5&#39;-CACCAGC-3&#39; (H13260) kimyasal 5&#39;- ve 3&#39;-fosfat ihtiva eden 2&#39;-O-metil RNA sentez edildi. Bu sgRNAlardır insan lösemi hücre hattı, HL60, ve bir insan miyeloma hücre hattı RPMI-8226 canlılığı üzerindeki etkileri incelendi. Hücre canlılığı deneylerinin Örnek neden olur. Şekil 1 &#39;de dört sgRNAlardır H1885, H3277, H10927 gösterilir ve H13260 çok etkili ve sırası ile&gt;% 80 ve&gt;% 70 ile HL60 ve RPMI-8226 hücre canlılığı azaltılmış. heptamerlerin H3277 ve H10927 de kanserli olmayan HEK293 hücre canlılığı üzerindeki etkileri bakımından test edildi, ve neredeyse Affe gösterilmiştirct hücre 22 canlılığı. Annexin V ve 7AAD çift boyama ile akış sitometrisi heptamer H1885, H3277, H10927 veya H13260 ile muamele HL60 hücreleri için gerçekleştirilmiştir. Her sgRNA, erken ve geç apoptotik aşamalarında toplam hücre sayısı (56-95%) sahte (Şekil 2) bu (% 4-6) daha yüksek oldu. Bu gözlemler, HL60 hücre canlılığının azaltılması apoptoza bağlı olduğuna işaret etmektedir. <img alt="Şekil 1" src="/files/ftp_upload/53879/53879fig1.jpg" /> Şekil 1. Hücre canlılığı ölçülür. Hücre canlılığı, HL60 hücrelerinde (A) veya RPMI-8226 hücreleri (B) yokluğunda ya da çıplak heptamer tipi sgRNA H1885 1 uM, H3277, H10927 mevcudiyetinde kültürlenmiştir 72 saat sonra ölçüldü H10944, H12287 veya H13260. sgRNAlardır yokluğunda göreceli canlı hücre sayıları 100 Hata ayarlanırçubuklar SD gösterir (n = 3). * P &lt;0.002. Lösemi Araştırma, Vol yayımlanmaktadır. 38. Takahashi, M. ve diğ., Hematolojik maligniteler karşı potansiyel tedavi aktif maddeleri, s 808-815, Şek bir heptamer tipi sgRNA kütüphanesinin taranması. 1, Telif Hakkı (2014), Elsevier izniyle. <a href="https://www.jove.com/files/ftp_upload/53879/53879fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="şekil 2" src="/files/ftp_upload/53879/53879fig2.jpg" /> Şekil 2. Akış sitometrisi. HL60 hücreleri ile ilgili olarak, akış sitometrisi Annexin V ve 7AAD çift boyama yapıldı. Hücreler yokluğunda ya da çıplak heptamer tipi sgRNA H1885 (A), H3277 (A) H13260 (A) ya da H10927 (B) 1 uM varlığında kültürlenen 72 saat sonra analiz edilmiştir. Lösemi Araştırma, Vol yayımlanmaktadır. 38, Takahashi, M. ve diğ., Hematolojik maligniteler karşı potansiyel tedavi aktif maddeleri, s 808-815, Şek bir heptamer tipi sgRNA kütüphanesinin taranması. 2, Telif Hakkı (2014), Elsevier izniyle. <a href="https://www.jove.com/files/ftp_upload/53879/53879fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents at JoVE.com

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

Burada kan kanserlerine karşı potansiyel terapötik ilaçların bir heptamer tipi sgRNA kitaplığının taranması için bir protokol mevcut.

DOĞRU gen susturma: Potansiyel Kanser Tedavi Ajanlar için heptamer tipi Küçük Rehberi RNA Kütüphanesi Taranması

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

true-gene-silencing-screening-heptamer-type-small-guide-rna-library

Nanyang Technological University

Research

JoVE Journal

Medicine

5.6K Görüntüleme.  Niigata University of Pharmacy and Applied Life Sciences.  Burada kan kanserlerine karşı potansiyel terapötik ilaçların bir heptamer tipi sgRNA kitaplığının taranması için bir protokol mevcut. 

Video: TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

Tedavi Oncolytic virusu modüle ana faktörleri tanımlamak için genom çapında RNAi tarama

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Kanser Araştırmaları

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

Kanser gen Fonksiyon Eğitim için DNA vektör tabanlı RNA Girişim

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

Tıp

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

HOX Loci odaklı CRISPR/sgRNA Kütüphane eleme tanımlayan kritik CTCF sınırları

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

Genetik

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Dizili Kütüphanelerin Orta İşleç Lik Taraması ve Çift Luciferase Tabanlı Muhabir kullanılarak Transkripsiyon Faktörü Düzenleyicilerin Tanımlanması

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Kültür Hücreler Kanser ile ilişkili Sinyal İletimi sorgulanması için bir Multiplexed Lusiferaz tabanlı Tarama Platformu

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

Bir İlaç Direnci Fenotipini Modüle Eden Faktörleri Tanımlamak için Havuzlanmış shRNA Kütüphanesi Taraması

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

Tümörlerde Terapötik Hedefleri Belirlemek ve Karşılaştırmak için Genom Ölçeğinde Tarama

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Yazar Spotlight: Genom Ölçekli shRNA Ekranları Aracılığıyla Malign Periferik Sinir Kılıfı Tümörü için Yeni Terapötik Hedefler Bulma 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

Malign Periferik Sinir Kılıfı Tümörünün Fare Modellerinde Terapötik Hedefleri Belirlemek için Genetik Profilleme ve Genom Ölçeğinde Bırakma Taraması

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

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.

Memeli Hücreleri RNAi Tarama MİSYON esiRNA

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 ...

DOĞRU gen susturma: Potansiyel Kanser Tedavi Ajanlar için heptamer tipi Küçük Rehberi RNA Kütüphanesi Taranması

Özet

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Tedavi Oncolytic virusu modüle ana faktörleri tanımlamak için genom çapında RNAi tarama

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HOX Loci odaklı CRISPR/sgRNA Kütüphane eleme tanımlayan kritik CTCF sınırları

Dizili Kütüphanelerin Orta İşleç Lik Taraması ve Çift Luciferase Tabanlı Muhabir kullanılarak Transkripsiyon Faktörü Düzenleyicilerin Tanımlanması

Kültür Hücreler Kanser ile ilişkili Sinyal İletimi sorgulanması için bir Multiplexed Lusiferaz tabanlı Tarama Platformu

Bir İlaç Direnci Fenotipini Modüle Eden Faktörleri Tanımlamak için Havuzlanmış shRNA Kütüphanesi Taraması

Malign Periferik Sinir Kılıfı Tümörünün Fare Modellerinde Terapötik Hedefleri Belirlemek için Genetik Profilleme ve Genom Ölçeğinde Bırakma Taraması

Malign Periferik Sinir Kılıfı Tümörünün Fare Modellerinde Terapötik Hedefleri Belirlemek için Genetik Profilleme ve Genom Ölçeğinde Bırakma Taraması

Malign Periferik Sinir Kılıfı Tümörünün Fare Modellerinde Terapötik Hedefleri Belirlemek için Genetik Profilleme ve Genom Ölçeğinde Bırakma Taraması

Memeli Hücreleri RNAi Tarama MİSYON esiRNA