저자는 마티아스 베드로와 이러한 방법이 개발되었다 취리히의 ETH에서 생화학 연구소에서 자신의 그룹을 감사합니다. 이 작품은 스위스 국립 과학 재단 (National Science Foundation)에 의해 지원되었다.

1. 현미경 측정 <ol><li> 효모와 합성 배지 5 ㎖를 접종한다. </li><li> 하룻밤 30 ° C에서 세포를 성장. </li><li> 다음날 아침 밤새 문화의 OD 600을 측정합니다. </li><li> 600 0.05 과다 복용 5 ㎖ 합성 배지에서 하룻밤 문화를 희석. </li><li> 최소 4 시간 동안 30 ° C에서 희석 문화를 성장. </li><li> 준비 3 배 합성 배지 (0.6 M) NaCl 용액 집중. </li><li> PBS에서 인 Concanavalin A (CONA)의 1 ㎎ / ㎖의 솔루션을 준비합니다. </li><li> 여과 액 ~ 잘 슬라이드에 CONA 용액 200 μL. </li><li> 30 분 동안 서 보자. </li><li> CONA 솔루션을 제거합니다. </li><li> 웰에 150 μL의 H 2 O를 추가합니다. </li><li> H 2 O를 제거 </li><li> 세포 배양의 OD 600을 측정합니다. </li><li> 1.5 ml의 마이크로 튜브에 = 0.02 OD 600에서 세포 배양의 300 μl를 준비합니다. </li><li> 45 초 동안 수욕에서 초음파 처리 셀. </li><li> 소용돌이 짧게 마일crotube. </li><li> 45 초 동안 수욕에서 초음파 처리 셀. </li><li> 간단히 소용돌이 모세관. </li><li> 우물에 세포 배양 200 μl를 추가합니다. </li><li> 세포가 우물의 바닥에 정착하도록 30 분을 기다립니다. </li><li> 현미경에 잘 슬라이드를 놓습니다. </li><li> 기록 될 채널에 대한 형광 조명 세팅을 선택한다. </li><li> (: 세포의 적절한 분할 수 있도록 초점면 아래의 3 μm의 약간 초점의 하나) 두 명 시야 이미지의 조명 설정을 선택합니다. </li><li> 시간 경과 측정 시간 간격을 선택 </li><li> 이미지 뷰의 필드를 선택합니다. </li><li> 몇 프레임의 인수를 시작합니다. </li><li> 0.6 M의 NaCl 매체의 100 μl를 추가 인수를 일시 중지합니다. </li><li> 영상을 다시 시작합니다. </li></ol> 2. 유동 세포 계측법 측정 <ol><li> 효모와 합성 배지 5 ㎖를 접종한다. </li><li> 하룻밤 30 ° C에서 성장. </li><li> 측정OD 하룻밤 문화의 600 다음날 아침. </li><li> 600 0.1 과다 복용 5 ㎖ 합성 배지에서 하룻밤 문화를 희석. </li><li> OD 0.2 ~ 0.4의 600에 도달하기 위해 최소 4 시간 동안 30 ° C에서 성장. </li><li> H 2 O에서 사이클로 헥시 미드 용액을 1 ㎎ / ㎖를 준비 </li><li> 준비 3 배 합성 배지 (0.6 M) NaCl 용액 집중. </li><li> 측정 할 때마다 포인트 0.6 M의 NaCl 매체의 100 μL와 하나의 1.5 ML 마이크로 튜브를 준비합니다. </li><li> 시간 0에서, 각각의 마이크로 튜브에 세포 배양 200 μl를 추가합니다. </li><li> 30 ℃에서 진탕 배양한다 </li><li> 시간 t에서, 마이크로 튜브에 30 μL의 사이클로 헥시 미드를 추가합니다. </li><li> 원하는 모든 시점에 2.11 단계를 반복합니다. </li><li> 적어도 45 분 동안 모든 마이크로 튜브를 품어 떨고와 30 ° C에서 최대 3 시간. </li><li> 400 ㎕의 PBS와 FACS 튜브를 준비합니다. </li><li> 1 분 동안 수욕에서 초음파 처리 셀. </li><li> 간단히 소용돌이 모세관. </li><li> 아들1 분 동안 수욕 세포 icate. </li><li> 간단히 소용돌이 모세관. </li><li> 각 모세관에서의 대응 FACS 튜브에 100 ㎕의 세포 배양을 추가합니다. </li><li> 검출을위한 488 nm의 여기 레이저와 30분의 530 nm의 대역 통과 필터를 선택합니다. </li><li> 테스트가 아닌 표현과 완전히 그들이 검출기 감도 범위에 빠지고 있는지 확인하기 위해 샘플을 표현. </li><li> 획득 한 각 샘플에 대한 10,000 세포를 측정한다. </li></ol>

<ol>
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B., Levine, A. J., Siggia, E. D., Swain, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+gene+expression+in+a+single+cell.">Stochastic gene expression in a single cell.</a> Science. 297 (5584), 1183-1186 (2002).</li><li>Pelet, S., Dechant, R., Lee, S. S., van Drogen, F., Peter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+image+analysis+platform+to+quantify+signal+transduction+in+single+cells.">An integrated image analysis platform to quantify signal transduction in single cells.</a> Integrative biology : quantitative biosciences from nano to macro. 4 (10), 1274-1282 (2012).</li><li>Carpenter, A. E., Jones, T. 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Chapter 14, Unit 14.18 (2008).</li><li>Dimopoulos, S., Mayer, C., Rudolf, F., Stelling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+Single+Cell+Segmentation+in+a+Variety+of+Microscopy+Images.">Automatic Single Cell Segmentation in a Variety of Microscopy Images.</a> Current protocols in molecular biology. , (2012).</li><li>Miyawaki, A., Nagai, T., Mizuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+protein+fluorophore+formation+and+engineering.">Mechanisms of protein fluorophore formation and engineering.</a> Current Opinion in Chemical Biology. 7 (5), 557-562 (2003).</li><li>Hersen, P., McClean, M. N., Mahadevan, L., Ramanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+processing+by+the+HOG+MAP+kinase+pathway.">Signal processing by the HOG MAP kinase pathway.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (20), 7165-7170 (2008).</li><li>Pepperkok, R., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+fluorescence+microscopy+for+systems+biology.">High-throughput fluorescence microscopy for systems biology.</a> Nature reviews Molecular cell biology. 7 (9), 690-696 (2006).</li></ol>

저자는 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

현미경 사용 CONA와 잘 치료는 세포의 적절한 이미징을 보장하는 필수 단계이다. CONA은 PBS (5 ㎎ / ㎖)에서 낮은 용해도를 갖기 때문에, 여과 처리 수는 여과되지 않은 용액에 존재하고 촬상 방해 큰 응집물의 제거. 세포는 처리 된 표면에 비교적 강하게 부착하고 유도 용액의 첨가는 자극 전후 지속적인 추적을 허용 세포의 현지화를 방해해서는 안된다. 세포는 정 유량 7,15을</...

전달 폭포를 통해 신호는 종종 단백질의 발현에 절정. 이 발현 프로파일의 특성화는 생물학적 경로의 기능을 이해하는데 중요한 요소이다. 위로 통제 단백질과 그들의 활성화의 역학의 스펙트럼의 식별은 마이크로 어레이, 북부 오 또는 서부 오 1-3와 같은 다양한 기술에 의해 달성 될 수있다. 그러나, 이러한 기술은 세포의 전체 모집단의 응답을 평균. 단백질 발현의 미세 조절을 이해하기 위해, 단일 세포 수준에서 측정을 수집하는 것이 바람직하다. 이상적으로, 이러한 측정은 기본 경로의 수학적 모델을 개발하는 의무가 정량적 데이터를 제공해야합니다. 현미경 및 유동 세포 계측법 이상적으로 이러한 양적 단일 세포 측정을 제공하기 위해 적합한 두 가지 기술이다. 형광 단백질과 단백질의 내생 태그는 B 수전자 발현 레벨 4를 정량화하는 데 사용됩니다. 단백질의 말단에 큰 형광 부분의 첨가는 비 기능을 렌더링 할 수 있기 때문에 그러나, 형광 구조체의 발현을 구동 프로모터에 따라 특정 식 기자를 생성하는 것이 더욱 바람직하다. 이 기자의 단백질은 셀룰러 시스템에 외생 적이며, 따라서 세포에서 일어나고있는 신호 이벤트에 영향을주지 않습니다. 현미경 및 유동 세포 계측법 모두 널리 효모 신호 분야에서 사용되어왔다. 예로서, 콜먼 - 러너와 동료는 상관 관계, 하나의 세포, 효모 짝짓기 신호 전달 캐스케이드 5의 노이즈를 정량화하기 위해 현미경으로 구체적이고 구조적으로 표현 기자 짝짓기의 표현, ACAR 및 규제 네트워크를 연구하는 유동 세포 계측법을 사용하는 동료 GAL 6 유전자의 발현을 제어한다. 이전 연구 7에서, 우리는 combinati을 사용했습니다높은 삼투압 글리세롤 효모 신진에서 (HOG) 경로에서 표현의 출력을 연구하는이 두 기술에. 이 미토 겐 활성화 단백질 키나제 (MAPK) 경로는 하이퍼 삼투압 스트레스에 의해 트리거된다. 그것은 약 300 유전자의 발현 결과 전사 프로그램을 유도하는 세포의 핵으로 옭 Hog1 MAPK의 활성화를 초래한다. 이 과정을 연구하기 위해, 우리는 배 금성 형광 단백질 (P의 STL1-QV)의 발현을 구동 STL1 프로모터 (Hog1 활동 8에 대한 응답으로 구체적으로 유도하는 유전자)를 기반으로 식 기자를 설계했다. 측정은 형광 기자를 표현하는 인구의 비율과 중간 스트레스 수준 (0.1 M의 NaCl)에서 세포의 두 집단의 존재를 발견 유동 세포 계측법. 우리는 더 이상이 문제를 조사하기 위해 현미경을 사용하고 단백질 발현이 노이즈가 고유 요인 (9)에 의해 지배 된 것을 발견했다. 우리는 기LD 더욱 Hog1 활동의 유사한 수준의 세포가 현저하게 상이한 발현 결과를 표시 할 수 있다는 것을 관찰한다. 이 두 기술의 조합은 우리가 스트레스 반응 유전자의 느린 리모델링이 하나의 셀 레벨 7의 발현 결과에 영향을 방법을 설명 할 수있었습니다. 이 논문에서, 우리는 현미경에 의한 단백질 발현의 정량의 예를 들어 하이퍼 삼투 충격에 의해 유도 된 P STL1-QV 기자의 표현을 사용하고 유동 세포 계측법. 0.2 M의 NaCl 스트레스 대상이 같은 변형은 두 가지 방법으로 연구 하였다. 이것은 우리가 이러한 상호 보완적인이 두 기술의 몇 가지 주요 차이점을 강조 할 수 있습니다.

<table border="1">
		<tbody>
		 <tr>
			 <td></td>
			 <td></td>
			 <td></td>
			 <td>Reagent</td>
		 </tr>
		 <tr>
			 <td>Yeast Nitrogen Base</td>
			 <td>ForMedium</td>
			 <td>CYN6202</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>CSM amino-acid mix</td>
			 <td>ForMedium</td>
			 <td>DCS0011</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Concanavalin A</td>
			 <td>GE Healthcare</td>
			 <td>17-0450-01</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Cycloheximide</td>
			 <td>Fluka Aldrich Sigma</td>
			 <td>C7698</td>
			 <td>Toxic</td>
		 </tr>
		 <tr>
			 <td>ySP9</td>
			 <td></td>
			 <td></td>
			 <td>W303Mata leu2::LEU2-pSTL1-quadrupleV enus</td>
		 </tr>
		 <tr>
			 <td>pSP34</td>
			 <td></td>
			 <td></td>
			 <td>pRS305 pSTL1 (-800 -0) - quadruple V enus</td>
		 </tr>
		 <tr>
			 <td></td>
			 <td></td>
			 <td></td>
			 <td>Material</td>
		 </tr>
		 <tr>
			 <td>Microscope</td>
			 <td>Nikon</td>
			 <td>Ti-Eclipse</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Microscope control software</td>
			 <td>Micro-manager</td>
			 <td>Ver 1.4.11</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Incubation chamber</td>
			 <td>LIS</td>
			 <td>The Box</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Fluorescence light source</td>
			 <td>Lumencor</td>
			 <td>SpectraX</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Camera</td>
			 <td>Hamamatsu</td>
			 <td>Flash 4.0</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Flow cytometer</td>
			 <td>BD</td>
			 <td>FACS calibur</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Sonicator bath</td>
			 <td>Telesonic</td>
			 <td>TUC-150</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>8-well-slide </td>
			 <td>Thermo Lab-tek</td>
			 <td>155409</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>96-well-plate</td>
			 <td>Matrical bioscience</td>
			 <td>MGB096-1-2-LG</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>FACS tube</td>
			 <td>BD Falcon</td>
			 <td>352054</td>
			 <td></td>
		 </tr>
		</tbody>
 </table>

temporal quantification of mapk induced expression in single yeast cells

단일 세포 수준에서의 유전자 발현의 정량화는 집단 수준에서 수행되는 측정에 가려진 신규 규제 메커니즘을 폭로. 두 가지 방법 현미경을 기반으로 유동 세포 계측법은 이러한 데이터를 취득 할 수있는 방법을 보여주기 위해 제공됩니다. 효모에서 고 삼투압 글리세롤 MAPK 경로의 활성화시에 유도 된 형광 리포터의 발현은 예로서 사용된다. 각 방법의 구체적인 장점은 강조 표시됩니다. 계측법 다수의 셀 (10000)을 측정하고 형광 단백질의 성숙 느린 동역학 독립적 단백질 발현의 역학의 직접적인 측정을 제공 흐른다. 현미경으로 살아있는 세포의 영상이 적은 세포에서 기자의 성숙 형태의 측정에 한정 대조적입니다. 그러나,이 기술에 의해 생성 된 데이터 세트는 여러 기자의 조합 및 시공간 정보에 매우 풍부 감사 될 수각각의 세포에서 얻은. 이 두 가지 측정 방법의 조합은 신호 전달 경로에 의해 단백질 발현의 조절에 대한 새로운 통찰력을 제공 할 수 있습니다.

현미경 사용 발현 리포터 P STL1-QV (ySP9 7) 베어링 효모 세포는 잘 슬라이드 하단에 부착되고, 현미경하에 넣었다. 세포 이미징 세션 도중에 직접 우물에 응력 매체의 첨가에 의해 자극 하였다. 이것은 우리가 통로 유도하기 전에 세포의 몇 가지 이미지를 획득하고 자극 후 자신의 운명을 수행 할 수 있습니다. 본 경우에, 세포를 10 분의 시간 간격으로 1 ~ 2 시간 동안...

Watch this Scientific Journal Video about Temporal Quantification of MAPK Induced Expression in Single Yeast Cells at JoVE.com

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

유동 세포 계측법 및 현미경에 따라 두 개의 보완적인 방법은 효모에서 MAPK 경로의 활성화에 의해 유도되는 유전자 발현의 역학의 단일 세포 수준에서 정량을, 가능하게하는 표시됩니다.

단일 효모 세포에서 MAPK 유도 표현의 시간적 정량화

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

temporal-quantification-mapk-induced-expression-single-yeast

Research

JoVE Journal

Biology

8.4K 조회수.  University of Lausanne.  유동 세포 계측법 및 현미경에 따라 두 개의 보완적인 방법은 효모에서 MAPK 경로의 활성화에 의해 유도되는 유전자 발현의 역학의 단일 세포 수준에서 정량을, 가능하게하는 표시됩니다. 

비디오: Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

우두 바이러스 감염 및 바이러스 유전자 발현의 시간적 분석 : 1 부

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

연구

생물학

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

우두 바이러스 감염 및 바이러스 유전자 발현의 시간적 분석 : 2 부

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

우두 바이러스 감염 및 바이러스 유전자 발현의 시간적 분석 : 제 3

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Methylotrophic 효모에서 재조합 단백질의 표현 Pichia pastoris의</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

성장 저해 Phenotypes의 확인은 효모에서 세균의 종류 III Effectors 표현에 의해 유도된

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

저산소증에 따라 단일 세포 수준에서 글로벌 RNA의 합성 분석

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

담배 과도 식 시스템을 사용하여 MAPK 신호 전달 통로의 공간 개편 이미징

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

마이크로 유체 장치를 사용하면 단일 신진 효모 세포의 수명과 세포 표현형을 측정하는

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...