화이트 헤드 연구소 펠로 자금

1 부 : 감염을 설정 <ol><li> flasks의 헬라 세포 성장과 세포가 약 80 % 합류 때까지 기다립니다. </li><li> 2퍼센트 FBS와 정기 DMEM없이 추가 항생제 : 실험에 대한 충분한 바이러스성 성장 매체를 준비합니다. </li><li> 감염이 중 원유 알려진 titer와 우두 바이러스 재고, 또는 당신이 호스트 유전자 발현이 염려되는 경우에는 알려​​진 titer와 바이러스를 정화 자당과 함께 수행할 수 있습니다. </li><li> 당신이 자당 정화 바이러스를 사용하는 경우, 2 부 직접 진행합니다. </li><li> 방금 전에 감염, 원유 우두 바이러스 주식을 사용하는 경우 대부분 얼음 목욕과 약간의 물과 컵 sonicator에 바이러스의 나누어지는을 sonicate. <ul style="list-style-type:lower-alpha;"><li> 20 초 동안 30W 주변에 Sonicate. </li><li> 소용돌이 튜브를 얼음과 추위로 가득한 컵을 유지하기 위해 필요할 경우 더 많은 얼음을 추가 sonication에게 3 번 반복합니다. </li><li> 각 sonication 단계 사이에 소용돌이. </li><li> 2 부 진행합니다. </li></ul></li><li> 또는, 당신이 컵 sonicator을 가지고하지 않으면 원유 바이러스 주식 및 0.25mg/mL의 트립신의 동일한 음량을 섞는다. <ul style="list-style-type:lower-alpha;"><li> 노골적인 소용돌이. </li><li> 30 분 37 ° C waterbath에 바이러스 재고 / 트립신 믹스를 품어. </li><li> 5-10분 간격으로 소용돌이. </li><li> 2 부 진행합니다. </li></ul></li></ol> 2 부 : 감염 세포 <ol><li> 감염의 원하는 다중성 (뫄)에서 monolayer를 감염하는 데 필요한 바이러스 입자의 양을 계산합니다. 일반적으로, (5 사이 10) 뫄 높은가 감염 timecourses에 사용됩니다. </li><li> 37 자당 정화 바이러스 또는 trypsinized 원유 바이러스의 원하는 금액을 추가 ° C 바이러스성 성장 매체와 잘 섞는다. 당신은 술병의 바닥을 감추기에 충분 할만큼 미디어를 사용해야합니다. 예를 들어, T - 175 플라스크 미디어 10 ML. </li><li> 세포에서 미디어를 제거하고 객실 온도 PBS로 씻어. </li><li> 각 플라스크에 바이러스 / 바이러스 성장 미디어를 추가합니다. <ul style="list-style-type:lower-alpha;"><li> 부드럽게 소용돌이, 37에서 틸트 접시 품어 ° 1 시간 동안 5 % CO2 배양기에서 C. </li><li> 기울기와 소용돌이 접시는 15 분 간격으로 균일하게 바이러스를 확산하고 세포 촉촉한 유지합니다. </li><li> 0hr/Mock의 시점 들어, 바이러스가 추가되지 않는 상태로, 오직 바이러스성 성장 미디어를 추가합니다. </li></ul></li><li> 1 시간 부화 후, 바이러스가 들어있는 미디어를 제거하고 객실 온도 PBS로 세 번 씻어. </li><li> 각 플라스크에 바이러스성 성장 매체의 최대 금액을 추가합니다. 예를 들어, T - 175 플라스크에 대한 미디어의 30 ML. 귀하의 공 인사 시점으로 이것을 계산 시작합니다. </li></ol> 파트 3 : 착취 세포 <ol><li> 현미경 세포를 확인하고 cytopathic 효과 (CPE)를 적어 둡니다. </li><li> 미디어를 제거하고 객실 온도 PBS의 30 ML로 세포를 씻어. </li><li> 세포에 트립신 15 ML을 추가하고 37 2 5 분 대한 품어 ° C. </li><li> 세포 분리에 대한 현미경으로 확인하고 세포를 이동시키다을 부드럽게 휴대용 술병을 누릅니다. </li><li> 50 ML 원뿔 관에 멸균 피펫 serological와 세포를 전송합니다. </li><li> 세포 성장 매체의 동일한 볼륨으로 술병을 세척하고, 원뿔 튜브 trypsinized 세포에 미디어를 추가합니다. </li><li> 상온에서 5 분 300 x g에서 세포를 원심 분리기. </li><li> 세포 펠렛에서 트립신 / 미디어를 제거합니다. </li><li> 원하는 RNA 격리 키트에서 TRIzol 시약 또는 용해 버퍼 중 하나의 세포 펠렛을 Resuspend. </li><li> 당신이 TRIzol를 사용하는 경우 -80에서 1.5mL 표시 Eppendorf 튜브 및 동결 1 ML aliquots에 샘플을 나누어 ° C. </li><li> 시점 당 하나의 휴대용 술병을 수확 모든 시간 지점 반복합니다. </li></ol> 4 부 : TRIzol에서 샘플 RNA 추출 <ol><li> 이 시점에서 당신의 샘플을 처리할 수 준비 TRIzol 시약에 resuspended하고 있어야합니다. </li><li> 각각의 튜브에 TRIzol의 모든 1 ML에 대한 BCP 단계 분리 시약의 200μL를 추가합니다. 당신은 TRIzol 많은 양의와 함께 시작하는 경우 큰 튜브에 샘플을 전송해야 할 수도 있습니다. </li><li> 소용돌이 또는 적극적으로 악수 후 2-3 분 동안 상온에서 알을 품다. </li><li> 15 분 동안 12,000 XG에서 샘플을 원심 분리기. </li><li> 새로운 튜브에 수성 단계를 전송합니다. 수성 단계 상단에있는 명확한 계층입니다. 당신은 당신이 밖으로 시작 TRIzol의 모든 1mL 약 600μL 회복해야합니다. </li><li> 대신 BCP의 두 번째 페놀 / 클로로포름 추출, 1 ML TRIzol 당 클로로포름의 500μL를 사용하여이 시간을 마십시오. </li><li> 소용돌이 또는 적극적으로 악수 후 2-3 분 동안 상온에서 알을 품다. </li><li> 15 분 동안 12,000 XG에서 샘플을 원심 분리기. </li><li> 새로운 튜브에 수성 단계를 전송합니다. 수성 단계 상단에있는 명확한 계층입니다. </li><li> 각 샘플에 선형 아크릴 아미드의 20μg을 추가합니다. 선형 아크릴 아미드는 항공사로서 역할을하고 RNA을 촉진하는 데 도움이됩니다. </li><li> 추가 500 &amp; MICRO, 이소프로판올의 L은 TRIzol 1 ML 당 당신은 각각의 샘플을 함께 시작 잘 섞는다. </li><li> 10 분 실온에서 샘플을 품어. </li><li> 10-15분에 대해 최대 속도 microcentrifuge에 원심 분리기. </li><li> RNA 펠렛은 튜브의 하단에 표시됩니다. 아주 조심스럽게, 진공 흡인기로하거나, 튜브의 표면에 뜨는를 제거하거나, 수동으로 pipettor로. 펠렛을 방해하지 마십시오. </li><li> 70 % 에탄올 1 ML로 펠렛을 씻으십시오. <ul style="list-style-type:lower-alpha;"><li> 펠렛에 70 % 에탄올 1 ML을 추가합니다. </li><li> 최고 속도 5-7분에 대한 14,000g을 원심 분리기. </li><li> 아주 조심스럽게, 진공 흡인기로하거나, 튜브에서 에탄올을 제거하거나, 수동으로 pipettor로. 펠렛은 튜브의 하단에 표시되었는지 확인합니다. </li><li> 뜨는 폐기하십시오. </li></ul></li><li> 세척 단계를 반복합니다. 뜨는이 삭제되면 펠렛은 튜브에 어디에, 마크. </li><li> 오분 이상 더 이상 공기 건조 펠릿. overdry하지 마십시오, 또는 RNA는 reconstitute하기 어려울 것입니다! </li><li> 이 예제에서 남아있는 DNA를 제거하는 옵션 DNase 처리를 수행하려는 경우 nuclease없는 물을 17μL의 펠렛을 resuspend. 그렇지 않으면, nuclease없는 물을 20μL의 펠렛을 resuspend 22 단계로 진행합니다. </li><li> (옵션 DNase 처리) Qiagen RNase 무료 DNase 세트를 사용하여 2μL 버퍼 RDD와 1μL가 resuspended RNA에 RNase 무료 DNase I을 재구성 추가합니다. 부드럽게 섞는다. </li><li> (옵션 DNase 처리) 37에서 품어 ° C를 30 분. </li><li> (옵션 DNase 처리) 65 2.5 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산)과 부화의 2μL 추가 ° 5 분 C는 DNase를 inactivate 수 있습니다. 더 이상 시간 / 높은 온도는 RNA의 저하를 일으킬 수 있으므로 불활 성화 시간 또는 온도를 초과하지 마십시오. </li><li> 분광 광도계에 의해 RNA 농도를 확인합니다. </li><li> -80 ° C.에서 RNA 샘플을 저장 </li><li> (선택 사항 QC) 애질런트 BioAnalyzer를 사용하여 RNA의 품질을 확인하거나, denaturing 젤에서 샘플을 실행 OD 판독을 선택하여. (260nm 및 280분의 260 비율) </li></ol>

<ol>
	<li>KH, R. u. b. i. n. s., LE, H. e. n. s. l. e. y., GW, B. e. l. l., Wang, C., EJ, L. e. f. k. o. w. i. t. z., PO, B. r. o. w. n., DA, R. e. l. m. a. n. <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., JM, G. a. t. e. l. l., 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>

중요 단계 파트 1 &amp; 2 동기 우두 감염을 설정하는 몇 가지 중요한 단계가 있습니다, 바이러스의 첫번째 목표가되어주의 sonication (또는 trypzinizing), 바이러스 입자를 disaggregate하기 위해서. 우두는 통합으로 높은 경향이 있으며, 바이러스 입자의 장애는 세포도 감염을 보장하는 것이 중요합니다. 동기 감염을 달성하기 위해, (2보다 큰) 뫄 높은 각 세포가 감염...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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		<tbody>
		
<tr>
<td>TRIzol Reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15596-026</td>
<td>Similar reagents, such as TriPure from Roche, will also work.</td>
</tr>
<tr>
<td>BCP Phase Separation Reagent</td>
<td>Reagent</td>
<td>Molecular Research Center</td>
<td>BP151</td>
<td>&nbsp;</td>
<tr>
<td>RNase-Free DNase Set</td>
<td>Reagent</td>
<td>Qiagen</td>
<td>79254</td>
<td>DNase treatment is an optional step.</td>
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vaccinia virus infection &amp; temporal analysis of virus gene expression: part 1

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 1 at JoVE.com

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

호스트 및 바이러스성 유전자 발현의 헬라 세포 및 분석의 우두 감염 프로토콜. 3 부 1.

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 ...

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10.2K 조회수.  MIT - Massachusetts Institute of Technology. 호스트 및 바이러스성 유전자 발현의 헬라 세포 및 분석의 우두 감염 프로토콜. 3 부 1.

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

Bacterial Gene Expression Analysis Using Microarrays

Microarrays을 사용하여 세균의 유전자 발현 분석

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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 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

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 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 ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

활성 T.의 체외의 Reconstitution에서 castaneum의 Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

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 ...