이 작품은 대상 중심의 R &amp; D, 일본 과학 기술 진흥기구, 일본의 사립 학교에 대한 홍보 및 공제 Corporation의 과학 연구 진흥 기금을 통해 적응력과 원활한 기술 이전 프로그램에서 지원하고, JSPS KAKENHI ​​그랜트 번호 24300342과 15H04313했다 .

헵 타머 형 1. 건설 sgRNA 도서관 <ol><li> 본격적인 라이브러리의 구축에 사용되지 않는 한, 384 (4-7) -7- NT 시퀀스의 서브 세트를 선택한다. 참고 : 순서는 랜덤 및 / 또는 특정 세포의 RNA를 대상으로 설계 할 수있다. 후자의 경우, 헤어핀 구조 -4,10-의 바로 하류 -7- NT 서열에 상보 적절한 컴퓨터 프로그램 및 / 또는 시각, 선택 시퀀스들을 이용하여 표적 RNA에서의 tRNA의 T 아암 닮은 헤어핀 구조를 찾아 17, 18. </li><li> 로 선택한 heptamers의 각을 합성 완전히 2&#39;- O -methylated, 5&#39;- 및 DNA / RNA 합성과 3&#39;- 인산화 RNA. 제어 된 기공 유리 (CPG) 지지체 상 포스 방법을 사용하여 5&#39;- 말단 4,4&#39;- 디메 톡시 트리 틸 (DMT) 합성의 마지막 사이클에서 (5 &#39;포스페이트를 추가) 보호기를 남겨 23. 참고 : 사용자 정의 한건데nthetic RNA는 뉴클레오티드 공급 업체에서 얻을 수 있습니다. </li><li> 마지막 합성주기의 완료 후, 8 시간 동안 55 ° C에서이를 1 ㎖의 29 % 암모늄 하이드 록 사이드를 추가 테플론 라이너 스크류 캡이있는 유리 병에 컬럼으로부터 합성 올리고 뉴클레오티드와 CPG를 전송하고, 부화 소비재에서 올리고 뉴클레오티드를 절단하고 기지와 인산염에서 보호 그룹을 제거합니다. </li><li> 40 ° C에서 원심 증발기로 암모늄 히드 록 시드를 증발시키고, 0.2 mL의 0.1 M N-hexylammonium 아세테이트로 올리고 뉴클레오티드를 재용. </li><li> 10~50% 아세토 니트릴 / 0.1 M N-hexylammonium 아세테이트를 함유하는 완충액, 폴리스티렌 - 디 비닐 벤젠 칼럼을 사용하는 역상 고성능 액체 크로마토 그래피를 통해 정제하여 합성 올리고 뉴클레오티드. 80 ° C에서 25 분 동안 / 2.0 ml의에서 분 열을 실행합니다. </li><li> 20 % 아세트산 용액을 제조하기 위해 분류 된 샘플 (1-2 ㎖)로 농축 아세트산 0.25-0.5 mL를 넣고 incubaTE, 실온에서 1 시간 동안이 용액은 DMT기를 제거한다. </li><li> 40 ° C에서 원심 증발기를 사용하여 샘​​플을 건조하고 1 ml의 29 % 암모늄 하이드 록 사이드에서 그것을 재 - 용해하고, 5 &#39;포스페이트의 측쇄를 제거하여 실온에서 15 분 동안 용액을 배양한다. </li><li> 40 ° C에서 원심 증발기로 약 0.3 ml의 시료의 부피 (1 ml)로 줄인다. </li><li> 차단 1000을 4 ℃에서 밤새 분자량 투석 튜브를 사용하여 증류수에 대한 전체 농축 샘플 (500 ml)에 투석. </li><li> 그 후, 피펫으로 1.5 ㎖의 튜브로 투석 튜브로 투석 샘플을 이동시켜 40 ℃에서 원심 증발기로 건조하고, 볼륨있는 경우, 물에 대한 주입 등급 물에 재용 더 큰 100 μM 솔루션을 만들기에 충분 작은. </li><li> 분광 광도계로 260 nm에서의 RNA 시료의 광학 밀도를 측정하고 (100)의 농도를 조정물에 대한 주입 등급의 물을 첨가하여 μM. -20 ° C 사용하기 전에 아래의 RNA 솔루션을 저장합니다. </li></ol> 세포 생존에 대한 sgRNA 도서관 2. 심사 <ol><li> 준비 103 세포 / 99 μL / 웰 (예 : HL60 및 RPMI-8226) 10 % 소 태아 혈청과 1 % 페니실린 - 스트렙토 마이신 용액이 보충 된 RPMI-1640 배지에서 96 웰 접시. 단지 배경 공제 미디어를 포함하는 우물을 준비합니다. 샘플 우물 주변 우물을 비우 멸균 물을 추가, 가장자리 효과를 제거합니다. </li><li> 각 웰에 물에 대한 주입 수준의 물에 용해 헵 타머 형 sgRNA (100 μM) 1 μl를 추가합니다. 세중 각 sgRNA를 분석. 3 일간 5 % CO 2 가습 배양기에서 37 ℃에서 플레이트를 인큐베이션. </li><li> 각 웰에 시판 WST-8 솔루션의 적절한 볼륨을 추가합니다. 1 내지 4 시간 동안 동일한 인큐베이터에서 37 ℃에서 플레이트를 인큐베이션. </li><li> 흡광도 A를 측정마이크로 플레이트 리더 450 nm의에서 t. 주 : 대부분의 경우, 흡광도 및 생존 세포 수 사이의 선형성 흡광도 값이 1.0를 초과하지 않는 한 유지 될 것으로 보인다. </li></ol> 유동 세포 계측법에 의한 효과적인 sgRNAs 3. 평가 <ol><li> 준비 103 세포 / 10 % 소 태아 혈청과 1 % 페니실린 - 스트렙토 마이신 용액이 보충 된 RPMI-1640 배지에서 96 웰 접시에 99 μL / 웰 (예 : HL60). 하나 sgRNA가 테스트 될 때까지 적어도 10 우물을 준비합니다. 샘플 우물 주변 우물을 비우 멸균 물을 추가, 가장자리 효과를 제거합니다. </li><li> 각 웰에 물에 대한 주입 수준의 물에 용해 헵 타머 형 sgRNA (100 μM) 1 μl를 추가합니다. 3 일간 5 % CO 2 가습 배양기에서 37 ℃에서 플레이트를 인큐베이션. </li><li> 피펫으로 5 ㎖의 튜브에 적어도 10 웰로부터 동일한 sgRNA로 처리 된 세포를 수집한다. 30 원심 분리기에서 5 분 동안 튜브를 스핀0 XG, 미디어를 폐기합니다. </li><li> 튜브로 1X 인산 완충 생리 식염수 (PBS) 2 mL를 넣고 세포를 다시 일시. 300 XG에 원심 분리기에서 5 분 동안 튜브를 스핀, 그리고 PBS를 폐기합니다. 튜브에 1 배 넥신 V 결합 버퍼의 150 μl를 추가하고 세포를 다시 일시 중지합니다. </li><li> , 피코 에리 트린의 2 μL (PE) π 공역 넥신 V 용액 튜브 내지 7 aminoactinomycin의 D (7AAD) 액 1 μl를 추가들을 잘 혼합하고, 어둠에서 실온에서 15 분 동안 튜브를 배양한다. (24) 플로우 사이토 미터를 사용하여 샘플을 분석한다. </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 du...

(TR Nase Z의 L 후 라 - 유 전자 fficacious 유전자 침묵을 tilizing) TRUE 유전자 침묵은 RNA 지시 유전자 침묵 기술 (1) 중 하나입니다. 이 기술은 개발 tRNase Z L (또는 3 &#39;tRNase), tRNA의 3&#39;처리 endoribonuclease의 속성을 기반으로하고있다 : 그것은을 인식하여 인공 작은 가이드 RNA (sgRNA)의 지시에 따라 어떤 예상 사이트에있는 모든 대상 RNA를 절단 할 수 있습니다 9 - 표적 RNA와 sgRNA (2) 사이에 형성된 프리 tRNA의 형상 또는 마이크로 사전의 tRNA 형 구조. 길이 NT를 일반적으로 7-31이다 sgRNA는 네 그룹, 5&#39;- 절반의 tRNA, 헵 타머 RNA, 14 NT를 선형 RNA, 그리고 훅 RNA로 분류된다. 15 - 우리는 셀 (10) 생활에 다양한 인위적으로 설계 sgRNAs을 도입하여 TRUE 유전자 침묵의 효과를 증명하고있다. 우리는 또한 sgRNAs이 세포 위스콘신에 의해 흡수 될 수 있음을 보여 주었다모든 형질 전환 시약 사전 통 보없이 그들의 표적 RNA 수준을 하향 조절 및 / 또는 인간 암세포 (16)에 세포 자멸을 유도 할 수있다 - 18. 21 - TRUE 유전자 침묵은 tRNase Z L과 자연 sgRNAs (19)를 통해 세포 내와 세포 간 유전자 조절 네트워크 시스템에서 작동하도록 나타납니다. 우리는 혈액 암 (22)에 대한 잠재적 인 치료 헵 타머 형 sgRNAs 검색 156 헵 타머 형 sgRNAs 함유 sgRNA 라이브러리를 구축했다. 우리는 인간 골수종 및 백혈병 세포의 생존에 대한 효과를 스크리닝하고 20 효율적으로 암세포의 적어도 하나의 세포 사멸을 유도 할 수 있음을 발견 하였다. 그 중 4 마우스 이종 이식 실험에서 종양 성장 속도를 감소시키는 것으로 나타났다. 여기에서 우리는 혈액 암에 대한 잠재적 인 치료 약물에 대한 헵 타머 형 sgRNA 라이브러리의 스크리닝을위한 프로토콜을 제시한다. 이 프로토콜은 어떻게 포함세포 생존, 어떻게 더 유동 세포 계측법에 의해 유효 sgRNAs을 평가하는 각 sgRNA의 효과를 평가하는 방법 sgRNA 라이브러리를 구성합니다. 이러한 프로토콜이 포함되지 않더라도 마우스 이종 이식 모델에서의 셀룰러 sgRNA 표적 RNA를위한 분석 및 sgRNAs 효과의 평가는, 17, 22 종래의 방법에 따라 수행 될 수있다.

<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

TRUE 유전자 침묵은 (TR Nase Z의 L 후 라 - 유 전자 fficacious 유전자 침묵 tilizing)의 tRNA 3 &#39;처리 endoribonuclease, tRNase Z L을 안내하는 인공 작은 가이드 RNA (sgRNA)을 이용 기술을 입을 RNA의 지시 유전자 중 하나입니다 , 표적 RNA를 인식한다. sgRNAs 모든 형질 전환 시약없이 세포에 의해 용해 될 수 있고, 그들의 표적 RNA 수준을 하향 조절 및 / 또는 인간의 암세포에서 아폽토시스를 유도 할 수있다. 우리는 인간 골수종 및 백혈병 세포의 생존에 미치는 영향 156 헵 타머 형 sgRNAs 함유 sgRNA 라이브러리를 스크리닝하고 20 효율적으로 암세포의 적어도 하나의 세포 사멸을 유도 할 수 있음을 발견 하였다. 여기에서 우리는 혈액 암에 대한 잠재적 인 치료 약물에 대한 헵 타머 형 sgRNA 라이브러리의 스크리닝을위한 프로토콜을 제시한다. 이 프로토콜은 세포 생존, 그리고 각 sgRNA의 효과를 평가하는 방법 쿵푸를하는 방법 sgRNA 라이브러리를 구성하는 방법을 포함rther는 유동 세포 계측법에 의해 유효 sgRNAs을 평가합니다. 약 2,000 명중은 16,384 heptamers 이루어지는 본격적인 sgRNA 라이브러리를 스크리닝함으로써 얻어 질 것으로 예상된다.

여섯 헵 타머 형 sgRNAs 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), 및 5&#39;-CACCAGC-3&#39;(H13260)을 화학적으로 3&#39;- 및 5&#39;- 포스페이트를 함유하는 O -2&#39;- 메틸 RNA 합성 하였다. 이러한 sgRNAs은 인간 백혈병 세포주, HL60과 인간 골수종 세포주, RPM...

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

여기에서 우리는 혈액 암에 대한 잠재적 인 치료 약물에 대한 헵 타머 형 sgRNA 라이브러리의 스크리닝을위한 프로토콜을 제시한다.

TRUE 유전자 음소거 : 잠재적 인 암 치료제에 대한 헵 타머 형 소형 가이드 RNA 라이브러리의 심사

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

Research

JoVE Journal

Medicine

5.6K 조회수.  Niigata University of Pharmacy and Applied Life Sciences.  여기에서 우리는 혈액 암에 대한 잠재적 인 치료 약물에 대한 헵 타머 형 sgRNA 라이브러리의 스크리닝을위한 프로토콜을 제시한다. 

비디오: 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.

RNAi 게놈 넓은 검사 Oncolytic 바이러스 치료 변조 호스트 요소를 식별 하

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

연구

암 연구학

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.

암 관련 유전자 기능을 연구하기 DNA를 벡터 기반의 RNA 간섭

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

의학

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 Focused CRISPR/sgRNA 라이브러리 심사 식별 중요 CTCF 경계

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

유전학

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.

배열된 라이브러리의 중간 처리량 스크리닝및 이중 루시퍼라제 기반 리포터를 이용한 전사 계수 조절기 식별

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.

배양 세포에서 암과 연관된 신호 전달을 심문위한 멀티 플렉스 루시 페라 기반 스크리닝 플랫폼

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).

약물 내성 표현형을 조절하는 인자를 식별하기 위한 풀링된 shRNA 라이브러리 스크리닝

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

종양의 치료 표적을 식별하고 비교하기 위한 게놈 규모 스크리닝

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.

저자 스포트라이트: 게놈 스케일 shRNA 스크리닝을 통한 악성 말초 신경초 종양에 대한 새로운 치료 표적 찾기 

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.

악성 말초 신경초 종양의 마우스 모델에서 치료 표적을 식별하기 위한 유전자 프로파일링 및 게놈 규모 드롭아웃 스크리닝

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.

포유 동물 세포에서 RNAi 심사를위한 선교 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 ...

TRUE 유전자 음소거 : 잠재적 인 암 치료제에 대한 헵 타머 형 소형 가이드 RNA 라이브러리의 심사

요약

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RNAi 게놈 넓은 검사 Oncolytic 바이러스 치료 변조 호스트 요소를 식별 하

암 관련 유전자 기능을 연구하기 DNA를 벡터 기반의 RNA 간섭

HOX Loci Focused CRISPR/sgRNA 라이브러리 심사 식별 중요 CTCF 경계

배열된 라이브러리의 중간 처리량 스크리닝및 이중 루시퍼라제 기반 리포터를 이용한 전사 계수 조절기 식별

배양 세포에서 암과 연관된 신호 전달을 심문위한 멀티 플렉스 루시 페라 기반 스크리닝 플랫폼

약물 내성 표현형을 조절하는 인자를 식별하기 위한 풀링된 shRNA 라이브러리 스크리닝

악성 말초 신경초 종양의 마우스 모델에서 치료 표적을 식별하기 위한 유전자 프로파일링 및 게놈 규모 드롭아웃 스크리닝

악성 말초 신경초 종양의 마우스 모델에서 치료 표적을 식별하기 위한 유전자 프로파일링 및 게놈 규모 드롭아웃 스크리닝

악성 말초 신경초 종양의 마우스 모델에서 치료 표적을 식별하기 위한 유전자 프로파일링 및 게놈 규모 드롭아웃 스크리닝

포유 동물 세포에서 RNAi 심사를위한 선교 esiRNA