Name: applying fluorescence resonance energy transfer (fret) to examine effector translocation efficiency by coxiella burnetii during sirna silencing
Uploaded: 2016-07-06T15:00:00.000+00:00
Duration: 10 min 29 s
Description: Watch this Scientific Journal Video about Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing at JoVE.com

This work was supported by National Health and Medical Research Council (NHMRC) grants (Grant ID 1062383 and 1063646) awarded to HJN. EAL is supported by an Australian Postgraduate Award.

The authors have nothing to disclose.

분비 시스템, 세균 이펙터 단백질의 숙주 세포의 세포질에서 이러한 시스템의 전송은 많은 병원성 박테리아가 복제 성 고유 한 틈새 내에서 감염을 확립 이용하는 중요한 독성 성분이다. 많은 연구 그룹의 초점은 세균 이펙터 호스트 단백질 및 숙주 세포에서이 경로 이펙터가 영향 사이의 상호 작용을 조사했다. 매우 제한된 조사있는 경우 호스트 단백질 성공 세균 이펙터 전위 필요한 될 가능성?...

Coxiella burnetii의는 인수 공통 인체 감염 Q 열병을 일으키는 독특한 세포 내 병원균이다. 이 질환은 증상이 혈청에서 종종 노출 한 후 심내막염 년으로 나타난다 생명을 위협하는 만성 감염 연장 임상 프레 젠 테이션의 광범위한 스펙트럼과 연결됩니다. 인간의 감염은 주로 반추 동물, 특히 젖소, 양, 염소 감염의 주요 저수지와 오염 된 에어로졸의 흡입을 통해 발생합니다. 이러한 동물 Coxiella 감염은 일반적으로 무증상이지만 감염 낙태를 트리거 할 수 있으며, 유체 출산 및 태반 내의 상당한 세균 부하는 한 로컬 환경을 오염시킬 수있다. 공중 보건과 농업 산업 모두 최근 네덜란드 2에서 발생한 Q 열병 발생이 관찰되었다에 엄청난 부담 등 오염의 예는 수 있습니다. 2007 년과 사이2010, Q 열병 4,000 통해 인간의 경우는 진단받은이 발발은 염소 농장 3의 중요한 오염에 연결되었다. 또한, Coxiella 인해 심각한 이환율과 사망률 (4)을 일으킬 수있는 박테리아와 필요한 낮은 전염성 선량의 환경 안정성에 미국 질병 통제 예방 센터에 의해 분류로, 잠재적 인 생물학적 무기입니다. Coxiella은 두 단계에 존재하는 천연 소스에서 고립 된 단계 I 생물은 매우 악성이며, 단계 II 생물이 높은 생체 내에서 감쇠된다. 예를 들어, Coxiella burnetii 나인 마일 단계 I 생물의 여러 체외 통로 후, 단계 II 박테리아가 절단 된 리포 폴리 사카 라이드의 결과로 돌이킬 수없는 염색체 삭제를 포함하는 제조 하였다 (LPS) 5. 이 균주, C. burnetii NMII는 조직 배양 모델 I, 위상 표현형 유사하고 안전한 모드를 제공한다연구진은 실험실 5 Coxiella의 발병 기전을 연구하기위한 리터. 최근 몇 년 동안 몇 가지 혁신이 빠르게 Coxiella 유전학의 분야를 전진했다. 특히, 무균 매체의 개발 (시트르산 시스테인 매체 산성화 - ACCM-2)는 모두 액체 및 고체 배지에 6,7 Coxiella의 무 세포 성장을 허용했다. 이것은 유도 유전자 발현 시스템, 셔틀 벡터 및 랜덤 트랜스포존 시스템 8-11 포함 Coxiella 가능한 유전 도구 직접 개선 결과. 가장 최근에, 대상 유전자의 불 활성화에 대한 두 가지 방법이 특정 독성 유전자 후보 (12)를 조사하기위한 방법을 포장, 개발되었다. 폐포 대 식세포에 의해 내재화 이어 Coxiella는 막 결합 구획 내에 높은 번호에 복제는 액포 (CCV)을 함유 Coxiella- 불린다. CCV는 호스트 세포 내 이입 매매 일이 필요합니다거친 초기와 후기 엔도 좀이 리소좀 유래 세포 기관 (13)에 성숙 할 때까지. 이 과정을 통해 상기 CCV 중 하나가 일시적으로 나타나거나 포함 액포와 연관되지만 Rab5, Rab7, CD63 및 LAMP-1 13-15에 한정되지 유지 숙주 요인을 취득한다. 숙주 세포 내에서 Coxiella의 복제는 완전한 기능 도트 / ICM 형 IVB 분비 시스템 (T4SS) 8,16,17에 전적으로 의존한다. 이 분비 시스템 ancestrally 공액 시스템과 관련된 여러 단백질 구조 및 박테리아 단백질을 제공하기 위해 모두 세균과 액포 막에 걸쳐 호스트 세포질 18에, 이펙터 불린다. Coxiella T4SS는 기능적으로 레지오넬라 뉴모 필라 (19, 20)의 잘 특징 유형 IVB 점 / ICM 분비 시스템과 매우 유사합니다. Coxiella이 리소좀 유래 산성에 도달 할 때까지 흥미롭게 T4SS 이후 이펙터 전위의 활성화가 발생하지 않는다세포 기관, 약 8 시간 게시물 감염 17,21. 현재까지 130여 점 / ICM 이펙터는 9,17,22-24를 확인되었습니다. 이 이펙터의 대부분은 가능성이 숙주 세포 내에서 Coxiella의 복제시 중요한 역할을; 단, 약간의 이펙터 기능 25-29 특성화되었다. 본 연구에서 우리는 숙주 세포의 세포질 내 β-lactamase의 활성을 통해 (이하 BLAM 기판이라 함) CCF2-AM FRET 기판 (도 1)의 절단에 의존하는 형광 기반 전위 분석을 이용한다. 관심의 유전자는 구성 적 발현을 제공하는 리포터 플라스미드에 β-lactamase를 (연기하는거야)-1 TEM 융합된다. BLAM 기판을 FRET 쌍을 형성하는 두 개의 형광 발색단 (쿠마린 및 플루오 레세 인)로 구성되어있다. 플루 오레 신 및 이펙터 전위의 부재하에 녹색 형광 발광의 FRET의 쿠마린의 여기 결과; 그러나, 만약 즐M 이펙터 융합 단백질은 호스트 세포질로 전좌하고, 얻어진 β-lactamase의 활성을 절단 여진 다음 청색 형광 방출을 생산 FRET 쌍을 분리 BLA​​M 기판의 β 락탐 고리. 이 전위 분석이 잘 C. 등 다양한 세포 내 및 세포 외 박테리아의 범위에서 이펙터 단백질을 식별하는 방식으로 입증되었다 burnetii, L. 뉴모 필라, L. longbeachae, 클라미디아 트라코마 티스, 장내 유해 E. 대장균, 살모넬라 균 및 브루셀라 17,30-35. Coxiella 이펙터 전위 특정 숙주 인자의 역할을 결정하기 위해 우리는 특히 작은 간섭 RNA의 (siRNA의), RNA 간섭으로 알려진 유전자 침묵을위한 확립 된 방법을 이용한다. 원래 예쁜 꼬마 선충에서 발견, RNA 간섭은 타고난 defe에 사용되는 보존 내생 세포 과정바이러스에 대한 NSE뿐만 아니라 유전자 조절 (36, 37). 서열 - 특이 적 siRNA를 바인딩 한 후 mRNA를 분해 특정 유전자 침묵 또는 최저 38 결과 (RNA 유도 침묵 복합체) RISC 통해 발생한다. 이 연구에서의 siRNA가 세포 내 이입 경로의 중요한 레귤레이터 두 호스트 단백질 Rab5A 및 Rab7A를 대상으로 하였다. 이펙터 전위에 Rab5A 및 Rab7A 입을의 영향 C.을 사용하여 확인 하였다 burnetii pBlaM-CBU0077. 것은 이미 Coxiella 17 도트 / ICM 분비 시스템 전좌 것으로 나타났다 CBU0077로 선택 하였다. siRNA의 유전자 사일런 여기 기재된 fluorescencebased 전위 분석 양쪽을 이용하여 우리는 Coxiella 이펙터 단백질의 전위에 숙주 인자의 역할을 설정하기 시작한다. 이 방법은 유사한 secretio 소유 모두 세포 내 및 세포 외 박테리아의 넓은 범위에 적용될 수있다이펙터 단백질의 전위에 대한 책임 N 시스템.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Citric acid</td><td>Sigma</td><td>C0759</td><td>ACCM-2 medium component</td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>S4641</td><td>ACCM-2 medium component</td></tr><tr><td>Potassium phosphate</td><td>Sigma</td><td>60218</td><td>ACCM-2 medium component</td></tr><tr><td>Magnesium chloride</td><td>Calbiochem</td><td>442611</td><td>ACCM-2 medium component</td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C5080</td><td>ACCM-2 medium component</td></tr><tr><td>Iron sulfate</td><td>Fisher</td><td>S93248</td><td>ACCM-2 medium component</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9625</td><td>ACCM-2 medium component</td></tr><tr><td>L-cysteine</td><td>Sigma</td><td>C6852</td><td>ACCM-2 medium component</td></tr><tr><td>Bacto-neopeptone</td><td>BD</td><td>211681</td><td>ACCM-2 medium component</td></tr><tr><td>Casamino acids</td><td>Fisher</td><td>BP1424</td><td>ACCM-2 medium component</td></tr><tr><td>Methyl beta cyclodextrin</td><td>Sigma</td><td>C4555</td><td>ACCM-2 medium component</td></tr><tr><td>RPMI + Glutamax</td><td>ThermoFisher Scientific</td><td>61870-036</td><td>ACCM-2 medium component</td></tr><tr><td>Chloramphenicol</td><td>Sigma</td><td>C0378</td><td>For bacterial culture</td></tr><tr><td>ON-TARGETplus Non-targeting Control Pool (OTP)</td><td>Dharmacon</td><td>D-001810-10-05</td><td>Non-targeting control</td></tr><tr><td>siGENOME Human PLK1 (5347) siRNA - SMARTpool</td><td>Dharmacon</td><td>M-003290-01-005</td><td>Causes cell death; measure of transfection efficiency</td></tr><tr><td>siGENOME Human RAB5A (5968) siRNA - SMARTpool</td><td>Dharmacon</td><td>M-004009-00-0005</td><td></td></tr><tr><td>siGENOME Human RAB7A (7879) siRNA - SMARTpool</td><td>Dharmacon</td><td>M-010388-00-0005</td><td></td></tr><tr><td>5x siRNA buffer</td><td>Dharmacon</td><td>B-002000-UB-100</td><td>Use sterile RNase-free water to dilute 5x siRNA buffer to 1x siRNA buffer</td></tr><tr><td>DharmaFECT-1 Transfection Reagent</td><td>Dharmacon</td><td>T-2001-01</td><td>For transfection</td></tr><tr><td>Opti-MEM + GlutaMAX</td><td>ThermoFisher Scientific</td><td>51985-034</td><td>Reduced-serum medium used for transfection</td></tr><tr><td>DMEM + GlutaMAX</td><td>ThermoFisher Scientific</td><td>10567-014</td><td>For cell culture and infection</td></tr><tr><td>Heat-inactivated fetal calf serum (FCS)</td><td>Thermo Scientific</td><td>SH30071.03</td><td>Can use alternate equivalent product</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td>For storage of Coxiella strain</td></tr><tr><td>PBS</td><td></td><td></td><td>For cell culture</td></tr><tr><td>0.05% Trypsin + EDTA</td><td>ThermoFisher Scientific</td><td>25300-054</td><td>For cell culture</td></tr><tr><td>dH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>For dilution of samples and standards for qPCR</td></tr><tr><td>Quick-gDNA Mini Prep</td><td>ZYMO Research</td><td>D3007</td><td>Can use alternate equivalent product to extract gDNA</td></tr><tr><td>SensiFAST SYBR No-ROX Kit</td><td>Bioline</td><td>BIO-98020</td><td>Can use alternate equivalent qPCR master mix product for qPCR reaction</td></tr><tr><td>LiveBLAzer FRET-B/G Loading kit with CCF2-AM</td><td>Invitrogen</td><td>K1032</td><td>For measurement of translocation using fluorescence and generation of the 6X loading solution (contains Solution A, B, C and DMSO)</td></tr><tr><td>Sodium hydroxide</td><td>Merck</td><td>106469</td><td>Probenicid solution component</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>71505</td><td>Probenicid solution component</td></tr><tr><td>di-Sodium hydrogen orthophosphate</td><td>Merck</td><td>106586</td><td>Probenicid solution component</td></tr><tr><td>Probenicid</td><td>Sigma</td><td>P8761</td><td>Probenicid solution component</td></tr><tr><td>DRAQ5 Fluorescent Probe Solution (5 mM)</td><td>ThermoFisher Scientific</td><td>62251</td><td>Nuclei stain to determine cell viablity. Use 1:4000 diluted in PBS.&amp;nbsp;</td></tr><tr><td>4% PFA (paraformaldehyde) solution in PBS</td><td>Sigma</td><td>P6148</td><td>Fixing solution for HeLa 229 cells</td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask with vented cap</td><td>Corning</td><td>430639</td><td>For growth of bacterial strain</td></tr><tr><td>96 Well Flat Clear bottom Black Polystyrene TC-Treated Microplates, Individually wrapped, with Lid, Sterile</td><td>Corning</td><td>3603</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask with vented cap</td><td>Corning</td><td>430641</td><td>For growth of HeLa 229 cells</td></tr><tr><td>175 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask with vented cap</td><td>Corning</td><td>431080</td><td>For growth of HeLa 229 cells</td></tr><tr><td>Haemocytometer</td><td></td><td></td><td>For quantification of HeLa 229 cells</td></tr><tr><td>Tear-A-Way 96 Well PCR plates</td><td>4titude</td><td>4ti-0750/TA</td><td>For qPCR reaction</td></tr><tr><td>8-Lid chain, flat</td><td>Sarstedt</td><td>65.989.002</td><td>For qPCR reaction</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bench top microfuge</td><td></td><td></td><td></td></tr><tr><td>Bench top vortex</td><td></td><td></td><td></td></tr><tr><td>Orbital mixer</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td>For pelleting bacterial culture</td></tr><tr><td>Nanodrop</td><td></td><td></td><td>For gDNA quantification</td></tr><tr><td>Mx3005P QPCR machine</td><td>Aligent Technologies</td><td>401456</td><td>For quantification of Coxiella genomes in 7 day culture</td></tr><tr><td>ClarioSTAR microplate reader</td><td>BMG LabTech</td><td></td><td>For measurement of fluorescence</td></tr></tbody></table>

applying fluorescence resonance energy transfer (fret) to examine effector translocation efficiency by coxiella burnetii during sirna silencing

Coxiella burnetii의, Q 발열의 원인 물질은 복제 성 틈새를 확립 타입 IV 도트 / ICM 분비 시스템에 의존하여 세포 내 병원체이다. 이펙터의 코호트는 호스트 프로세스를 조작 및 복제에 대한 고유 리소좀 유래 액포의 확립을 허용하는 숙주 세포에 본 시스템을 통해 전좌된다. 특정 유전자의 siRNA를 이용하여 침묵과 β-lactamase의 활성에 의존하는 FRET 기반의 기판을 사용하여 이펙터 전위 측정 : 여기에 제시된 방법은 두 개의 잘 확립 된 기술의 조합을 포함한다. 이 두 접근 방법의 적용, 우리는 박테리아 분비 시스템 기능 및 이펙터 전위에서 호스트 인자의 역할을 이해하기 시작할 수있다. 이 연구에서 우리는 Rab5A과 Rab7A, 모두 세포 내 이입 인신 매매 경로의 중요한 규제 기관의 역할을 조사했다. 우리는 이펙터 translocatio의 감소 중 단백질 결과의 발현을 침묵 입증N 효율. 이러한 방법은 또한 쉽게 분비 시스템을 이용하는 다른 세포 및 세포 외 병원체를 조사하도록 변형 될 수있다. 이러한 방식으로, 세균 이펙터 전위에 관련된 호스트 요인 글로벌 영상이 드러날 수있다.

이 연구를 들면, C. CBU0077 이전 Coxiella 도트 / ICM 분비 시스템 (17)의 전좌 이펙터로 도시 된 바와 같이 burnetii pBlaM-CBU0077 균주를 선택 하였다. 앞서 감염, 칠일 C.의 게놈 / ㎖의 총 개수 burnetii pBlaM-CBU0077 배양 물을 사용 qPCR에 열거 하였다. (4)는 순환 액의 예를 보여주는 도표 (TR) 값은 다음의 두 qPCR에 표준 샘?...

Watch this Scientific Journal Video about Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing at JoVE.com

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

세균성 병원균과 호스트 사이의 상호 작용을 조사하는 것은 생물학적 연구의 중요한 영역입니다. 여기서는 BLAM 기판을 사용하여 siRNA의 유전자 사일런 동안 Coxiella burnetii에 의해 이펙터 전위를 측정하기 위해 필요한 기술들을 설명한다.

적용 형광 공명 에너지 전달 (FRET)을하여 이펙터 전좌 효율을 검사합니다<em&gt; Coxiella burnetii의</em&gt; siRNA를 음소거시

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

applying-fluorescence-resonance-energy-transfer-fret-to-examine

Research

JoVE Journal

Immunology and Infection

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

10.6K 조회수.  University of Melbourne.  세균성 병원균과 호스트 사이의 상호 작용을 조사하는 것은 생물학적 연구의 중요한 영역입니다. 여기서는 BLAM 기판을 사용하여 siRNA의 유전자 사일런 동안 Coxiella burnetii에 의해 이펙터 전위를 측정하기 위해 필요한 기술들을 설명한다. 

비디오: Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

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

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

식물 병원균의 분비 이펙터에서 RNA 침묵 억제제의 스크리닝 및 식별

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

유전학

Enforced Activation of Enhancer RNAs In Situ through the dCas9 Synergistic Activation Mediator System

Enhancers are pivotal genomic elements scattered through the mammalian genome and dictate tissue-specific gene expression programs. Increasing evidence has shown that enhancers not only provide DNA binding motifs for transcription factors (TFs) but also generate non-coding RNAs that are referred to as eRNAs. Studies have demonstrated that eRNA transcripts can play significant roles in gene regulation in both physiology and disease. Commonly used methods to investigate the function of eRNAs are constrained to &#8220;loss-of-function&#8221; approaches by knockdown of eRNAs, or by chemical inhibition of the enhancer transcription. There has not been a robust method to conduct &#8220;gain-of-function&#8221; studies of eRNAs to mimic specific disease conditions such as human cancer, where eRNAs are often overexpressed. Here, we introduce a method for precisely and robustly activating eRNAs for functional interrogation of their roles by applying the dCas9 mediated Synergistic Activation Mediators (SAM) system. We present the entire workflow of eRNA activation, from the selection of eRNAs, the design of gRNAs to the validation of eRNA activation by RT-qPCR. This method represents a unique approach to study the roles of a particular eRNA in gene regulation and disease development. In addition, this system can be employed for unbiased CRISPR screening to identify phenotype-driving eRNA targets in the context of a specific disease.

<meta charset="utf8"></head><body>dCas9 Synergistic Activation Mediator System을 통한 Enhancer RNAs In Situ의 강제 활성화

Enhancer RNAs (eRNAs) are non-coding RNAs produced from active enhancers. An optimal approach to study eRNA functions is to manipulate their levels in the native chromatin regions. Here we introduce a robust system for eRNA studies by using CRISPR-dCas9-fused transcriptional activators to induce the expression of eRNAs of interest.

dCas9 Synergistic Activation Mediator System을 통한 Enhancer RNAs In Situ의 강제 활성화

Enhancers are pivotal genomic elements scattered through the mammalian genome and dictate tissue-specific gene expression programs. Increasing evidence ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

단일 분자 물고기에 의한 전사 활성 알레일의 단일 세포 분석

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

생화학

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

세대와의 멀티 표현형 높은 콘텐츠 상영<em&gt; Coxiella burnetii의</em&gt; 트랜스포존 돌연변이

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

면역학 및 감염병학

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 ...

생물학

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

siRNA의 중재 진 입을와 결합 라이브 세포 칼슘 영상은 CA를 나타냅니다 2 + ER 막의 누출 채널과 규제 메커니즘

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

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

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

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

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

의 정량 및 자동화된 고속 처리량 게놈 전체의 RNAi 화면 C. elegans</em

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

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

적용 형광 공명 에너지 전달 (FRET)을하여 이펙터 전좌 효율을 검사합니다

요약

더 많은 비디오 탐색

이 비디오의 챕터

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

식물 병원균의 분비 이펙터에서 RNA 침묵 억제제의 스크리닝 및 식별

dCas9 Synergistic Activation Mediator System을 통한 Enhancer RNAs In Situ의 강제 활성화

dCas9 Synergistic Activation Mediator System을 통한 Enhancer RNAs In Situ의 강제 활성화

단일 분자 물고기에 의한 전사 활성 알레일의 단일 세포 분석

세대와의 멀티 표현형 높은 콘텐츠 상영

포유 동물 세포에서 RNAi 심사를위한 선교 esiRNA

siRNA의 중재 진 입을와 결합 라이브 세포 칼슘 영상은 CA를 나타냅니다^{2 +} ER 막의 누출 채널과 규제 메커니즘

의 정량 및 자동화된 고속 처리량 게놈 전체의 RNAi 화면 C. elegans

In Vitro (시험관 ) 표적 후성유전학적 침묵을 위한 엔지니어링된 전사 억제제 선택