우리는 원고에 대한 그녀의 귀중한 통찰력에 대한 박사 패트리샤 Manosalva 감사합니다. 기금은 국립 과학 재단 (National Science Foundation)의 식물 게놈 프로그램, 보너스 번호 DBI - 0605059에 의해 제공되었다.

1 부 : 식물 입을 사용 N. benthamiana 공장은 2 주위해야 ½ 주 된 어느 cotyledons하고 처음 2 시간 - 4 사실 단풍이 등장했습니다. 팔일 게시물 출현, 진정한 단풍이 아직 등장하지 않은 - 토마토 (솔라넘 lycopersicum) 식물 7을 사용하고 있습니다. 2 부 : VIGS 1 일 <ol><li> 각 실험, pTRV1을 품고 Agrobacterium tumefaciens의, pTRV2, pTRV2 - 자료실 및 pTRV2 - 호스트 대상 유전자 50 μg / kanamycin의 ML 100 μg / rifampicin의 ML과 보충 LB 한천 플레이트에 재배하고 있습니다. rifampicin이 Agrobacterium 줘서 않지만 kanamycin은 pTRV 플라스미드를위한 선택합니다. 2 일 30 ° C에서 번호판을 품어. PDS의 입을 것은 photobleach 수있는 식물을 일으킬 것이며, 입을 효율성 컨트롤로 사용됩니다. 또한, downregulated하는 유전자는 pTRV2 벡터로 복제해야합니다. 이 복제를 용이하게 수있는 게이트웨이 호환 pTRV2 벡터이며 리우 외 의해 설명되어 있습니다. (2002). </li></ol> 3 일 <ol><li> 변종 각 위에서 언급한 항생제와 LB 3 ML 액체 문화 - 2의 예방. 18시간 200 RPM - 16 30 ° C에서 흔들어으로 품어 </li></ol> DAY 4 <ol><li> kanamycin, rifampicin 200 μm의의 acetosyringone (표 1, 2, 3) 보조 액체 유도 미디어 (IM) 메신저 문화에 가장 큰 문화의 25 희석 : 1의 예방. acetosyringone은 IM이 환경에게 호스트 apoplast이 병원체 조우를 모방하면서 공장 구에 T - DNA 전송에 필요한 Agrobacterium의 비르 유전자의 유도로 사용됩니다. 20 30 ° C에서 흔들어에 의해 부화 - 24 시간 200 RPM을 </li></ol> 5 일 <ol><li> 3000 X G. 10 분 centrifugating하여 세포를 수확 원래 문화 10 MM MgCl 2, 10 MM MES 산도 5.5했던 동일한 볼륨에 Resuspend. 전지는 부드럽게 그들을 resuspend하는 vortexed 수 있습니다. </li><li> 3000 X G. 10 분 다시 전지를 Centrifugue 10 MM MgCl2, 10 MM MES 산도 5.5 원래의 문화의 절반 볼륨 Resuspend. </li><li> 각 세균성 문화 OD 0.3 600과 세균 현탁액을 준비합니다. pTRV1 문화 400 μm의의 최종 농도 acetosyringone 추가합니다. </li><li> pTRV1와 1-1 비율 pTRV2 (또는 관심있는 유전자를 포함하는 pTRV2)이있는 문화를 섞는다. 또한이 pTRV2 - PDS 컨트롤을 포함합니다. 최종 acetosyringone 농도 지금 200 μm의하고 각 문화가 0.15의 OD 600에서입니다 점에 유의하시기 바랍니다. </li><li> 침묵하는 유전자와 실험의 날짜와 침투 수 묘목을 라벨. </li><li> 바늘로 침투하는 각각의 나뭇잎에 구멍을 포크. 묘목에 박테리아 현탁액에 침투 1 ML 불필요한 주사기를 사용합니다. 토마토의 경우, 두 cotyledons 침투 N.위한 동안 benthamiana, 가장 큰이 진정한 나뭇잎을 침투. 각 세균 혼합물의 다섯 ML 15 N. 침투 정도로해야합니다 benthamiana 25 토마토 모종. infiltrations 사이 접종 후 다음날까지 식물에 물을하지 않음으로써 장갑을 변경하여 교차 오염을 피한다. </li><li> 식물은 20 유지하고있다 - 22 ° C, 16 시간 하루 길이들은 assays에 사용할 수 있습니다 전에 적어도 3 ½ 주에 대한 RH 50 % 성장 챔버 인치 </li></ol> 파트 3 : 대표 결과 그림 1은 N.있는 대표 실험을 보여줍니다 benthamiana, 토마토 식물 자료실에 대해 침묵. 식물은 carotenoids의 저하 금액과 식물의 관찰 특성 photobleaching 표현형을 보여줍니다. PDS - 침묵 제어 식물 들어, photobleaching 시작 마자 1로 볼 수 ½ 주 침투 후. <img src="/files/ftp_upload/1292/Figure 1A 1292.jpg" alt="그림 1A" /><img src="/files/ftp_upload/1292/Figure 1B 1292.jpg" alt="그림 1B" /> 그림 1. PDS 제어 유전자의 입을은 N. benthamiana (A), 토마토 (B) 공장에서 photobleaching됩니다. 사진 3 ½ 주 입을 후에 찍은. 표 1. 유도 매체 (IM)의 작성. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 400 ML </td><td> 소주 H 2 O </td></tr><tr><td> 4.88 g </td><td> MES (2 - (4 morpholino) 에탄 술폰산) </td></tr><tr><td> 2.5 g </td><td> 포도당 </td></tr><tr><td> 0.12 g </td><td> 아니 2 PO 4 </td></tr></tbody></table> DH 2 O와 475 ML의 최종 볼륨을 지참하고 5.6으로 산도를 조정합니다. 압력솥. 매체 식었 후, 20X AB 소금의 25 ML를 추가합니다. 표 2. AB 소금의 작성. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 20g </td><td> NH 4 Club 호텔 </td></tr><tr><td> 6g </td><td> MgSO 4 명이 7H 2 O </td></tr><tr><td> 3g </td><td> KCl </td></tr><tr><td> 0.2 g </td><td> CaCl 2 </td></tr><tr><td> 0.05 g </td><td> FeSO 4 명이 7H 2 O </td></tr></tbody></table> 증류수 1 리터의 최종 볼륨을 가져와. 압력솥. AB의 소금이 오렌지색 분말로 침전하지 않을 수도 있습니다. 그들의 사용하기 전에 소용돌이에 의해 그것들을 잘 섞는다. 표 3. 200 MM의 Acetosyringone의 작성. acetosyringone 그것이 사용됩니다 하루를 준비한다고 유의하시기 바랍니다. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 19.6 MG </td><td> Acetosyringone (3 &#39;, 5&#39; - Dimethoxy - 4&#39; - hydroxyacetophenone) </td></tr><tr><td> 500 μL </td><td> DMSO (디메틸 sulfoxide) </td></tr></tbody></table>

<ol>
	<li>Voinnet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+as+a+plant+immune+system+against+viruses.">RNA silencing as a plant immune system against viruses.</a> Trends Genet. 17, 449-459 (2001).</li><li>Carrington, J. C., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+microRNAs+in+plant+and+animal+development.">Role of microRNAs in plant and animal development.</a> Science. 301, 336-338 (2003).</li><li>Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microRNA+as+a+translational+repressor+of+APETALA2+in+Arabidopsis+flower+development.">A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development.</a> Science. 303, 2022-2025 (2004).</li><li>Palatnik, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+leaf+morphogenesis+by+microRNAs.">Control of leaf morphogenesis by microRNAs.</a> Nature. 425, 257-263 (2003).</li><li>Brigneti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+pathogenicity+determinants+are+suppressors+of+transgene+silencing+in+Nicotiana+benthamiana.">Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.</a> EMBO J. 17, 6739-6746 (1998).</li><li>Liu, Y., Schiff, M., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing+in+tomato.">Virus-induced gene silencing in tomato.</a> Plant Journal. 31, 777-786 (2002).</li><li>MacFarlane, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+the+tobraviruses.">Molecular biology of the tobraviruses.</a> J. Gen. Virol. 80, 2799-2807 (1999).</li><li>Senthil-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+study+to+determine+the+extent+of+gene+silencing+in+Nicotiana+benthamiana+and+other+Solanaceae+species+when+heterologous+gene+sequences+are+used+for+virus-induced+gene+silencing.">A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing.</a> New Phytol. 176, 782-791 (2007).</li><li>McCullen, C. A., Binns, 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=Agrobacterium+tumefaciens+and+plant+cell+interactions+and+activities+required+for+interkingdom+macromolecular+transfer.">Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer.</a> Annu. Rev. Cell Dev. Biol. 22, 101-127 (2006).</li><li>Rosebrock, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bacterial+E3+ubiquitin+ligase+targets+a+host+protein+kinase+to+disrupt+plant+immunity.">A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity.</a> Nature. 448, 370-374 (2007).</li><li>Miki, D., Itoh, R., Shimamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+of+single+and+multiple+members+in+a+gene+family+of+rice.">RNA silencing of single and multiple members in a gene family of rice.</a> Plant Physiol. 138, 1903-1913 (2005).</li><li>Voinnet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use,+tolerance+and+avoidance+of+amplified+RNA+silencing+by+plants.">Use, tolerance and avoidance of amplified RNA silencing by plants.</a> Trends Plant Sci. 13, 317-328 (2008).</li><li>Ekengren, S. K., Liu, Y., Schiff, M., Kumar-Dinesh, S. P., Martin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+MAPK+cascades,+NPR1,+and+TGA+transcription+factors+play+a+role+in+Pto-mediated+disease+resistance+in+tomato.">Two MAPK cascades, NPR1, and TGA transcription factors play a role in Pto-mediated disease resistance in tomato.</a> Plant J. 36, 905-917 (2003).</li><li>Liu, E., Page, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+cDNA+libraries+for+virus-induced+gene+silencing+(VIGS)+using+tobacco+rattle+virus.">Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus.</a> Plant. Methods. 4, 5-5 (2008).</li></ol>

바이러스 유발 유전자 입을은 빠른 반대로 유전자 화면을 허용하는 방법입니다. 그것은 T - DNA 또는 Arabidopsis와 옥수수 같은 특정 식물에서만 사용할 수 있습니다 transposon의 중재 유전자 노크 아웃의 생성을 방지합니다. 또한 공장 변환의 시간이 소요되는 과정을 circumvents하고 그들이 충분한 상동 10 또는 다른 호스트 대상 시퀀스가 입을 벡터 6, 11 창출 배열되어있다는 제공 ...

virus-induced gene silencing (vigs) in nicotiana benthamiana and tomato

RNA 간섭 (RNAi)는 dsRNA 1 트리거 높은 특정 유전자 입을 현상입니다. microRNAs, 비 단백질 코딩 유전자와 짧은 간섭 RNAS (siRNAs)에서 생산이 입을 메커니즘은 RNA의 규제의 두 가지 주요 클래스를 사용합니다. 식물은 transposons을 제어와 같은 꽃 기관 형성과 잎 개발 2,3,4으로 개발 프로세스를 통해 꽉 컨트롤을 발휘하는 RNAi를 사용합니다. 식물은 또한 바이러스에 의해 감염으로부터 자신을 방어하기 위해 RNAi를 사용합니다. 따라서, 많은 바이러스는 숙주 5 그들의 성공적인 식민지를 허용하도록 입을 유전자의 진압을 발전시켜 왔습니다. 바이러스 유발 유전자 입을 (VIGS)는 공장 RNAi - 중재 항바이러스 방어 메커니즘을 활용하는 방법입니다. 수정되지 않은 바이러스에 감염된 식물의 메커니즘은 특히 바이러스 게놈에 대해 타겟팅됩니다. 그러나, 호스트 유전자에서 파생 시퀀스를 들고 바이러스 벡터와 프로세스가 추가로 해당 호스트 mRNAs에 대해 타겟팅할 수 있습니다. VIGS이 플라스미드는 티, 전체 또는 입을를 대상으로 유전자 시퀀스의 일부를지고 재조합 바이러스를 통해 제공하는 식물 병원균 Agrobacterium tumefaciens의를 사용하여 공장에서 높은 처리량 기능 유전체학에 맞게 조정될 수 있습니다. 체계적 바이러스 확산과 내생 식물 RNAi 기계는 알아서. 대상 유전자에 해당하는 dsRNAs의 길이는 21-24 세포핵의 siRNAs로 ribonuclease의 주사위 놀이를하는 사람에 의해 만들어진 후 죽습 있습니다. 이 siRNAs는 궁극적으로 목표 성적 증명서 2 타락하는 RNA 유발 입을 복합 (RISC)를 안내입니다. 다른 벡터는 VIGS에 고용되어 있고 가장 자주 사용 중 하나가 담배 텅 바이러스 (TRV)를 기반으로합니다. TRV는 bipartite 바이러스 등, 두 개의 다른 A.로 tumefaciens의 변종은 VIGS에 사용됩니다. 하나는 pTRV2, 다른 하나는 코트의 단백질과 VIGS 6,7에 사용되는 순서를 항구 동안 복제 및 운동 바이러스 기능을 인코딩 pTRV1을 수행합니다. Nicotiana benthamiana과 유전자 입을의 변종 결과 모두의 혼합물과 함께 토마토 모종의 접종. photobleaching 원인 내생 phytoene desaturase (PDS) 유전자,의 입을는 VIGS 효율을위한 컨트롤로 사용됩니다. 그것은 지적되어야하지만, 토마토에 입을는 보통 N.보다 덜 효율적이다 benthamiana. 관심 유전자의 RNA 대본 풍부한는 항상 대상 유전자를 효율적으로 다운 - 규제되었음을 보장하기 위해 측정해야합니다. N.에서 그럼에도 불구하고, heterologous 유전자 시퀀스 benthamiana는 토마토와 반대 8 해당 orthologs을 침묵하는 데 사용할 수 있습니다.

Watch this Scientific Journal Video about Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato at JoVE.com

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

노크를 다운 유전자 표현의에 대한 바이러스 유도된 유전자 입을 (VIGS) 메서드에 대한 설명<em&gt; Nicotiana benthamiana</em&gt;, 토마토.

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 ...

virus-induced-gene-silencing-vigs-in-nicotiana-benthamiana-and-tomato

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Biology

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

52.4K 조회수.  Cornell University. 노크를 다운 유전자 표현의에 대한 바이러스 유도된 유전자 입을 (VIGS) 메서드에 대한 설명 Nicotiana benthamiana, 토마토.

비디오: Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

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 ...

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생물학

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

세포 배양의 유도와 Hypoxia의 시험

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

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 ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Ribosome 바운드 초기 Polypeptides의 분리<em&gt; 체외에서</em&gt; mRNA 함께 Translational 일시 중지 사이트를 식별하려면

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Ribosome 바운드 Polypeptides을 파악하는 도구로 SecM 체포 시퀀스를 사용하여

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

감자와 니코티아나 벤타미안의 농아침투 및 PVX 농병감염

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

최적화 및 활용<em&gt; 아그로 박테 리움</em에서&gt; 매개 과도 단백질 생산<em&gt;는 담배</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

질병 벡터 모기 유충의 키토산 / 간섭 RNA 나노 입자 중재 유전자 음소거

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

siRNA를 매개 음소거로 마우스 난 모세포의 감수 스핀들 평가

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Luciferase 보완성 이미징 분석 결과 Nicotiana benthamiana 잎 뚜렷이 결정 단백질 단백질 상호 작용 역동성에 대 한

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...