화이트 헤드 연구소 펠로 자금

1 부 : RNA로부터 cDNA 합성 <ol><li> 앰비온 아미노 알릴 MessageAmp II 키트를 사용하기 전에, 세척 버퍼에 100 % 에탄올의 권장 볼륨을 추가합니다. </li><li> PCR 반응 관에서 T7 oligo (DT) 프라이머 총 RNA 및 1μl의 5μg을 100ng 사이를 추가합니다. nuclease 무료 물로 12μl에 볼륨을 가져와. </li><li> 70 샘플을 품어 ° C thermocycler 10 분. </li><li> ° C와 원심 분리기 간략 70 RNA 샘플을 제거합니다. 얼음 놓습니다. </li><li> 1 일 스트랜드 합성 마스터 믹스를 준비하고 (오류 pipetting 10 % 없더군요)를 실온에서 보관. (표 1) </li><li> 부드럽게 피펫 마스터 믹스이나 제스처는 혼합 후 간단히 원심 분리기. </li><li> 각 RNA 샘플을 마스터 믹스 8μl을 전송합니다. 3-4 회를 아래와 pipetting하여 섞는다. </li><li> thermocycler 2 시간 동안 42 ° C에서 알을 품다. </li><li> 얼음 원심 분리기 샘플의 잠시 장소. 즉시 dsDNA 합성의 다음 단계로 진행합니다. </li><li> 얼음 2 차 스트랜드 합성 마스터 믹스 (오류를 pipetting 10 % 없더군요 포함). (표 2)를 준비 </li><li> 부드럽게 피펫 마스터 믹스이나 제스처는 혼합 후 간단히 원심 분리기. </li><li> 각 샘플에 80μl를 전송하고 부드럽게 3-4 회를 아래와 pipetting하여 섞는다. </li><li> thermocycler 2 시간 동안 16 ° C에서 알을 품다. </li><li> 2 시간 부화 후 cDNA 정리 단계로 진행 또는 -20 ° C. 즉시 동결 </li></ol> 2 부 : 두 번 좌초 cDNA 청소 <ol><li> 냉장고에서 cDNA 순수를 제거하고 사용하기 전에 30 분 방 온도로 평형 수 있습니다. 완전히 사용하기 전에 자기 cDNA 바인딩 구슬을 resuspend 수있는 병을 흔들어. </li><li> 1.5mL 튜브와 50-60에서 품어 ° C 이전의 2 시간 배양 기간 동안 적어도 10 분 동안에 나누어지는 nuclease없는 물. </li><li> 각 샘플에 cDNA 순수의 180μl를 추가하고, 철저하게 최대 pipetting 아래로 섞는다. 96 - 잘 왕복 하단 플레이트에 샘플을 전송합니다. </li><li> 부드럽게 최소한 2 분 동안 궤도 쉐이크에 대한 번호판을 흔들어하여 샘플을 섞어 계속합니다. </li><li> 자기 구슬을 캡처하는 자기 서서 접시를 이동합니다. 약 6 분 동안 증언대에 접시를 남겨 또는 혼합물이 투명하게되고 바인딩 비즈가 pelleted 때까지. </li><li> 조심스럽게 자기 구슬을 방해하지 않고 진공 흡인기로 뜨는을 대기음. 또는, 조심스럽게 피펫으로 뜨는을 제거하고 표면에 뜨는 폐기하십시오. </li><li> 자석 스탠드에서 플레이트를 제거합니다. </li><li> 각 잘하는 150μl cDNA 씻으 버퍼를 추가하고 중간 속도로 궤도 쉐이크에서 1 분 접시를 흔들. 구슬은 세척 버퍼의 낮은 표면 장력으로 인해,이 단계에서 분산 않을 것입니다. </li><li> 자기 구슬을 캡처하는 자기 서서 접시를 이동합니다. </li><li> 조심스럽게 자기 구슬을 방해하지 않고 진공 흡인기로 뜨는을 대기음. 또는, 조심스럽게 피펫으로 뜨는을 제거하고 표면에 뜨는 폐기하십시오. </li><li> 자석 스탠드에서 플레이트를 제거합니다. </li><li> 세차에게 150μl cDNA 씻으 버퍼와 함께 2 차 시간을 반복합니다. </li><li> 2 차 세척 후, 최대 속도로 궤도 쉐이크 2 분 동안 접시를 흔들어하여 비즈를 건조. overdry하지 마! </li><li> 각 샘플에 preheated nuclease없는 물 18μl를 추가하여 비즈에서 cDNA를 Elute. </li><li> 적극적으로 궤도 쉐이크 3 분 동안 접시를 흔들 다음 자성 비즈가 완전히 분산되어 있는지 확인합니다. 그렇지 않다면, 계속 떨고. </li><li> 자기 비즈가 완전히 분산이되면, 자기 구슬을 캡처하는 자기 서서 접시를 이동합니다. </li><li> 조심스럽게 (~ 16μl) eluted cDNA는 새로운 PCR 플레이트 (또는 PCR 튜브)로 전송할 수 있습니다. </li><li> 다음 단계로 바로 진행, 또는 -20 ° C.에서 cDNA를 동결 </li></ol> 제 3 부 : 시험 관내 전사의 (IVT) <ol><li> 상온에서 IVT 마스터 섞는다. (표 3) 준비 </li><li> 부드럽게 마스터 믹스 또는 혼합에 영화를 피펫, 그리고 짧게 원심 분리기. </li><li> 각 샘플에 24μl에 추가하고, 부드럽게 3-4 회를 아래와 pipetting하여 섞는다. </li><li> thermocycler 14 시간 동안 37 ° C에서 품어, 다음 단계에 대한 ° C까지 준비 4 만요. </li></ol> 4 부 : 아르나 청소 IVT 후 <ol><li> 소용돌이 RNA 바인딩 비즈 간단히 사용하기 전에도 혼합물을 얻을 수 있습니다. </li><li> 상온에서 아르나 바인딩 믹스를 준비 (표 4) 이것은 미리 할 수 있습니다 -. 준비 바인딩 조합은 최대 일주일 위해 상온에서 저장할 수 있습니다. </li><li> vortexing하여 잘 섞는다. </li><li> 적어도 10 분 동안 50-60 ° C에서 1.5mL 튜브와 부화로 나누어지는 아르나의 용출 버퍼. </li><li> 아르나 바인딩 믹스 70μl를 추가각 샘플과 3-4 회 위아래로 pipetting과로 잘 섞는다. </li><li> PCR 플레이트에서 96 잘 왕복 하단 플레이트에 샘플을 전송합니다. </li><li> 각 샘플에 이소프로판올 100 % 50μl를 추가하고 3-4 회 위아래로 pipetting과로 잘 섞는다. </li><li> 부드럽게 철저하게 샘플을 혼합하기 위해 최소한 2 분 동안 궤도 쉐이크에 접시를 흔들. </li><li> 자기 구슬을 캡처하는 자기 서서 접시를 이동합니다. 혼합물이 투명하게되고 바인딩 비즈가 pelleted 때까지 법정에 접시를 남겨주세요. </li><li> 조심스럽게 자기 구슬을 방해하지 않고 진공 흡인기로 뜨는을 대기음. 또는, 조심스럽게 피펫으로 뜨는을 제거하고 표면에 뜨는 폐기하십시오. </li><li> 자석 스탠드에서 플레이트를 제거합니다. </li><li> 각 잘하는 100μl 아르나 와시 솔루션을 추가하고 중간 속도로 궤도 쉐이크에서 1 분 접시를 흔들. 구슬 완전히이 단계에서 분산되지 않을 수 있습니다. </li><li> 자기 구슬을 캡처하는 자기 서서 접시를 이동합니다. </li><li> 조심스럽게 자기 구슬을 방해하지 않고 진공 흡인기로 뜨는을 대기음. 또는, 조심스럽게 피펫으로 뜨는을 제거하고 표면에 뜨는 폐기하십시오. </li><li> 자석 스탠드에서 플레이트를 제거합니다. </li><li> 세차에게 100μl 아르나 와시 솔루션 2 차 시간을 반복합니다. </li><li> 2 차 세척 후, 최대 속도로 궤도 쉐이크 1 분에 대한 번호판을 흔들어하여 비즈를 건조. 샘플을 overdry하지 마! </li><li> 각 샘플에 40μl preheated 아르나의 용출 버퍼를 추가하여 비즈에서 아르나을 Elute. </li><li> 강력 3 분 궤도 쉐이크에 번호판을 흔들 다음 마그네틱 비즈가 완전히 분산되어 있는지 확인합니다. 그렇지 않다면, 계속 떨고. </li><li> 자기 비즈가 완전히 분산이되면, 자기 구슬을 캡처하는 자기 서서 접시를 이동합니다. 뜨는은 청소 접속 아미노 알릴 통합의 아르나가 포함되어 있습니다. </li><li> 조심스럽게 새로운 PCR 플레이트 (또는 PCR 튜브)로 eluted 아르나를 전송합니다. </li><li> (선택 단계) NanoDrop 분광 광도계에 1.5μl를 측정하여 시료의 RNA 농도를 확인합니다. </li><li> 바로 다음 단계에 계속하거나, -80에서 아르나를 저장 ° C. </li></ol> 표 1 : cDNA 1 일 스트랜드 합성 마스터 믹스 <table border="1" cellpadding="8"><tbody><tr><td> 시약 </td><td> 한 반응에 대한 금액 </td></tr><tr><td> 10X 먼저 스트랜드 버퍼 </td><td> 2 μl </td></tr><tr><td> dNTP 믹스 </td><td> 4 μl </td></tr><tr><td> Ribonuclease 억제제 </td><td> 1 μl </td></tr><tr><td> 배열 스크립트 </td><td> 1 μl </td></tr></tbody></table> 표 2 : cDNA 2 차 스트랜드 합성 마스터 믹스 <table border="1" cellpadding="8"><tbody><tr><td> 시약 </td><td> 한 반응에 대한 금액 </td></tr><tr><td> Nuclease 무료로 물 </td><td> 63 μl </td></tr><tr><td> 10X 둘째 스트랜드 버퍼 </td><td> 10 μl </td></tr><tr><td> dNTP 믹스 </td><td> 4 μl </td></tr><tr><td> DNA 효소 </td><td> 2 μl </td></tr><tr><td> RNase H </td><td> 1 μl </td></tr></tbody></table> 표 3 : IVT 마스터 믹스 <table border="1" cellpadding="8"><tbody><tr><td> 시약 </td><td> 한 반응에 대한 금액 </td></tr><tr><td> aaUTP 솔루션 (75 ㎜) </td><td> 2 μl </td></tr><tr><td> ATP, CTP, GTP 믹스 (25 ㎜) </td><td> 12 μl </td></tr><tr><td> T7 UTP 솔루션 (75 ㎜) </td><td> 2 μl </td></tr><tr><td> T7 10X 반응 완충액 </td><td> 4 μl </td></tr><tr><td> T7 효소 믹스 </td><td> 4 μl </td></tr></tbody></table> 표 4 : 아르나 바인딩 믹스 <table border="1" cellpadding="8"><tbody><tr><td> 시약 </td><td> 한 반응에 대한 금액 </td></tr><tr><td> RNA 바인딩 비즈 * </td><td> 10 μl </td></tr><tr><td> 비드 Resuspension 솔루션 * </td><td> 4 μl </td></tr><tr><td> 백퍼센트 이소프로판올 ** </td><td> 6 μl </td></tr><tr><td> 아르나 바인딩 버퍼 컨센트레이트 </td><td> 50 μl </td></tr></tbody></table> * 먼저 비드 resuspension 솔루션 RNA 바인딩 비즈를 섞습니다. ** 이소프로판올를 추가하고 아르나 바인딩 버퍼 집중을 추가하기 전에 잘 섞는다.

<ol>
	<li>Rubins, K. H., Hensley, L. E., Bell, G. W., Wang, C., Lefkowitz, E. J., Brown, P. O., Relman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+viral+gene+expression+programs+during+poxvirus+infection:+a+transcriptional+map+of+the+vaccinia+and+monkeypox+genomes.">Comparative analysis of viral gene expression programs during poxvirus infection: a transcriptional map of the vaccinia and monkeypox genomes.</a> PLoS ONE. 3 (7), e2628-e2628 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+a+complete+poxvirus+transcriptome+reveals+an+immediate-early+class+of+genes.">Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes.</a> Proc. Natl. Acad. Sci. 105 (6), 2140-2145 (2008).</li><li>Satheshkumar, P. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poxvirus+transcriptome+analysis.">Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E62-E62 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A.+Reply+to+Satheshkumar+and+Moss:+Poxvirus+transcriptome+analysis.">A. Reply to Satheshkumar and Moss: Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E63-E64 (2008).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., Mu&#241;oz, M., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+gene+expression+survey+of+vaccinia+virus+infection+of+human+HeLa+cells.">Cellular gene expression survey of vaccinia virus infection of human HeLa cells.</a> J Virol. 77 (11), 6493-6506 (2003).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Conde, R., Pascual-Montano, A., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+reveals+characteristic+changes+of+host+cell+gene+expression+in+response+to+attenuated+modified+vaccinia+virus+Ankara+infection+of+human+HeLa+cells.">Microarray analysis reveals characteristic changes of host cell gene expression in response to attenuated modified vaccinia virus Ankara infection of human HeLa cells.</a> J Virol. 78 (11), 5820-5824 (2004).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., N&#225;jera, J. L., Zaballos, A., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+response+to+the+attenuated+poxvirus+vector+NYVAC:+upregulation+of+apoptotic+genes+and+NF-kappaB-responsive+genes+in+infected+HeLa+cells.">Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.</a> J Virol. 80 (2), 985-998 (2006).</li><li>Guerra, S., N&#225;jera, J. L., Gonz&#225;lez, J. M., L&#243;pez-Fern&#225;ndez, L. A., Climent, N., Gatell, J. M., Gallart, T., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+gene+expression+profiling+after+infection+of+immature+human+monocyte-derived+dendritic+cells+by+the+attenuated+poxvirus+vectors+MVA+and+NYVAC.">Distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors MVA and NYVAC.</a> 81 (16), 8707-8721 (2007).</li></ol>

The authors have nothing to disclose.

중요 단계 배열 데이터에 편견이 관찰되었습니다로서 증폭의 두 번째 라운드는 권장되지 않습니다. 각 효소 단계 (1 일 및 2 차 가닥 cDNA 합성, IVT)에서 신중하게 혼합하는 것은 각 단계의 효소 배양은 적절한 온도는 좋은 증폭 수율을 얻는 것이 중요합니다. 도 편차를 공기 하이브 리다이 제이션 오븐이나 물 목욕 하이브 리다이 제이션에서 IVT 중 2-3 도까지처럼...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Amino Allyl MessageAmp II aRNA amplification kit</td>
<td>Reagent</td>
<td>Applied Biosystems</td>
<td>AM1753</td>
<td>For 20 reactions</td>
</tr>
<tr>
<td>Amino Allyl MessageAmp II aRNA amplification kit</td>
<td>Reagent</td>
<td>Applied Biosystems</td>
<td>AM1821</td>
<td>For 100 reactions, in a 96-well format</td>
</tr>
<tr>
<td>NanoDrop ND-1000 UV-VIS spectrophotometer</td>
<td>Other</td>
<td>NanoDrop</td>
<td>ND-1000</td>
<td>Or equivalent spectrophotometer</td>
</tr>		</tbody>
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vaccinia virus infection &amp; temporal analysis of virus gene expression: part 2

가족<em&gt; Poxviridae</em&gt; 감염된 세포의 세포질에서 독점적으로 복제 바이러스를 포함하는 대형 더블 - 좌초된 DNA로 구성되어 있습니다. 회원<em&gt; orthopox</em&gt; 속 포함 천연두, 인간 작은 수두의 원인이되는 대리인, monkeypox, 그리고 우두 (VAC), 바이러스 제품군의 prototypic 회원. 비교적 큰 (~ 2백킬로바이트) 우두 게놈 내에서 유전자의 세 클래스가 인코딩됩니다 : 초기, 중간, 그리고 늦었어요. 세 클래스가 virally 인코딩 RNA의 polymerases에 의해 베꼈는데 있지만, 각 클래스는 바이러스의 생활주기에서 다른 기능을 제공합니다. Poxviruses 감염 동안에 호스트 세포 환경의 변조에 대해 여러 전략을 활용합니다. 두 호스트 및 바이러스 유전자 발현의 조절을 이해하기 위해서, 우리는 바이러스와 호스트 세포 모두에서 사본의 풍요로움을 분석하는 게놈 차원의 접근법을 활용했습니다. 여기서 우리는 몇 시간이 지점 후 감염에 우두 바이러스와 샘플링 RNA와 헬라 세포의 시간 코스 감염을 보여줍니다. 두 호스트 및 바이러스 총 RNA는 격리 및 유전자 발현 분석을 위해 microarrays에 하이브 리다이 제이션에 대한 증폭됩니다.

Watch this Scientific Journal Video about Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2 at JoVE.com

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

호스트 및 바이러스성 유전자 발현의 헬라 세포 및 분석의 우두 감염 프로토콜. 3 부 2.

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

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

vaccinia-virus-infection-temporal-analysis-virus-gene-expression-part

Research

JoVE Journal

Biology

7.9K 조회수.  MIT - Massachusetts Institute of Technology. 호스트 및 바이러스성 유전자 발현의 헬라 세포 및 분석의 우두 감염 프로토콜. 3 부 2.

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

Bacterial Gene Expression Analysis Using Microarrays

Microarrays을 사용하여 세균의 유전자 발현 분석

연구

생물학

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

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 were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

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

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

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

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

바이러스 유도 진 입을 (VIGS)에 Nicotiana benthamiana 토마토

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

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

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 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

커튼에서 Agrobacterium - 중재 바이러스 유발 유전자 입을 분석

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBL에서 MultiBac 단백질 복잡한 생산 플랫폼

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

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 ...