Biz Dr minnettar. SP Dinesh Kumar ve Yule Liu TRV VIGS vektörler için, Dr. Kenerley Chuck, Terry Wheeler, Jim Starr ve pamuk tohumu sağlayan Bayer CropScience. Bu çalışma PH NSF LS ve NIH tarafından finanse edildi

1. Grow pamuk fideleri <ol><li> Upland pamuk (Gossypium hirsutum) çeşitli Fibermax 832, Phytogen 425RF, Phytogen 480WR ve Deltapine 90 Metro Mix 700 (SunGR, Beavile, WA) içeren tencere (çapı 7 cm) tohumları Sow. </li><li> Bir büyüme odasında 12 saat light/12 saat karanlık fotoperiyodun ° C, 120 μE m -2 G -1 ışık, 23 plastik bir kubbe ile örtülü bir tepsi tencere tutun. </li><li> Kubbenin iki kotiledonlarında ortaya çıkarın. </li><li> Yaklaşık iki hafta sonra, VIGS tayini için iki tam genişletilmiş kotiledonlarında bitkiler kullanın. Bu aşamada, gerçek yaprakları henüz (Şekil 1) ortaya çıkmış değil. </li></ol> 2. GrCLA1 ​​geni taşıyan Construct VIGS vektör <ol><li> G. cDNA kütüphanesi PCR primerleri GrCLA1-F ile 5&#39;-GCCCTTTGTGCATCTTC-3 &#39;ve GrCLA1-R, 5&#39;-CTCTAGGGGCATTGAAG-3&#39; pamuk Cloroplastos alterados 1 geni (GrCLA1) Amplify raimondii yaprak dokuları. </li><li> EcoRI ve KpnI GrCLA1, PCR ürünleri Digest ve ligasyonu ile pYL156 (pTRV-RNA2) vektör takın. </li><li> 50 mg / Kanamisin ml içeren LB agar plaklarına klonlar Ekran. Restriksiyon enzimi sindirim ve sıralama yapıları olun. </li><li> Elektroporasyon Agrobacterium tumefaciens GV3101 plazmid Transform ve 28 LB sıvı ortamda kurtarmak ° C LB ile plakalar transformants seçin + Kanamisin (50 mg / ml) + Gentamisin (25 mg / ml). Bakteri, -80 ° C ile uzun süreli kullanım için% 25 gliserol içerisinde saklanabilir. </li></ol> 3. VIGS aşılama gerçekleştirin <ol><li> Üç gün önce taşıma pYL192 (TRV) Agrobacterium tumefaciens aşılama, çizgi dondurulmuş gliserol stokları RNA1, pYL156 (TRV): RNA2 (vektör tek başına) ve pYL156 (TRV): 50 mg / Kanamisin ml içeren LB agar plaklarına RNA2-GrCLA1 ve 25 mikrogram / mL Gentamisin. Plakalar için 28 ° C&#39;de 24 saat inkübe edin. </li><li> İki gün önce VIGS infiltrasyon, yukarıdaki plakalardan her inşa için tek bir koloni almak ve içine 5 ml, 50 mg / Kanamisin mL ve 25 mcg / ml Gentamisin ile desteklenmiş LB orta aşılamak; 28 bakteri kültürü büyütün ° C 50 rpm hızında bir rulo davul geceleme. </li><li> 50 ml LB orta (- (4 morfolino)-etan, propan sülfonik asit 2) ve 20 mcM acetosyringone 50 mcg / Kanamisin mL ve 25 mcg / mL Gentamisin, artı 10 mM (MES) ile desteklenmiş bir şişesi için yukarıdaki kültür aktarın. 28 ° C&#39;de 50 rpm hızında bir çalkalayıcı geceleme kültür büyütün. </li><li> Ertesi gün, 5 dakika 4000 rpm&#39;de agro-bakteriyel hücreler aşağı doğru döndürün; 10 mM MgCl 2, 10 mM (MES) ve 200 mcM acetosyringone içeren infiltrasyon tampon kültür yeniden askıya. 1.5 kültürü OD 600 ayarlayın. </li><li> 3 saat boyunca oda sıcaklığında tezgah kültür bırakın. </li><li> Agrobacterial infiltrasyon önce yumruk kotiledonlarında delip olmadan 25 G iğne ile pamuk bitkileri kotiledonlarında alt. Kotiledon her bölümünde (Şekil 2) Bir veya iki delik (doku yumuşaklık bağlıdır) delikli edildi. </li><li> 1:1 oranında RNA2 GrCLA1; el karışımı 1 kullanarak yaralama siteleri aracılığıyla kotiledonlarında alt sızmak: RNA1 ve pYL156 (TRV): RNA2 veya pYL156 (TRV) pYL192 Agrobacterial kültür süspansiyon (TRV) karıştırın ml iğnesiz şırınga (Şekil 3). </li><li> Bitkilerin plastik bir kubbe ile örtün ve loş ışık koşulu altında gece boyunca oda sıcaklığında sızmış bitkiler bırakın. </li><li> Bitkilerin büyüme oda sıcaklığı 23 transfer ° C, 120 μE m -2 S 12 saat 12 saat ışık / karanlık döngüsünü -1 ışık. </li></ol> Not: bitkiler, 23 ° C veya cam sera (25-28 ° C) ya altında VIGS çalışır. (23 - 25 ° C) sıcaklığın nispeten düşük olduğunda, aşılama için daha kolay ve fenotip ile karşılaştırıldığında daha yüksek (28-30 ° C) ya da dalgalanma sıcaklık üniforma daha fazla. Bir kubbe ile bitkileri kapsayan VIGS daha verimli hale getirir. <ol start="10"><li> 7 ~ 8 gün infiltrasyon susturma fenotip inceleyin. PYL156 (TRV) tarafından susturuldu bitkilerin gerçek yaprakları: RNA2-GrCLA1 ​​albino fenotip (Şekil 4) görüntülemek için başladı. Agrobacteria taşıma pYL156 (TRV) ile infiltre bitki: RNA2 kontrol olarak hizmet vermektedir. </li><li> Kontrolü ve susturulması pamuk bitkileri izole edilen RNA ile ters transkripsiyon polimeraz zincir reaksiyonu (RT-PCR) kullanarak endojen genlerin ekspresyon seviyesi inceleyerek gen susturulması verimliliği olun. </li></ol> Not: TRV geniş bir konak yelpazesine sahiptir ve bildirilmesi gereken bir patojen olarak VIGS-ed bitki karantina koşulları ve bitkilerin altında tutulmalıdır düzgün otoklavlanabilir ve deneyleri sonra imha edilmelidir. 4.Temsilcisi Sonuçlar: Agrobacterial karışımı ile el-infiltrasyon yaklaşık iki hafta sonra GrCLA1 ​​susturulması kaynaklanan albino fenotip açıkça gerçek yaprakları gözlendi. Susturulması verimliliği 50&#39;den fazla tesisleri ile birden fazla deneylerde hemen hemen% 100 ulaşır. Susturuldu bitkilerin yaprakları yaşlı yapraklar (Şekil 4) yeni bir mozaik ile gelişmekte olan bir düzgün dağıtılmış ve çok güçlü bir albino fenotipi gösterir. <img src="/files/ftp_upload/2938/2938fig1.jpg" alt="Şekil 1" /> Şekil 1 Agrobacterial infiltrasyon için kullanılan iki tam genişletilmiş kotiledonlarında iki hafta eski aşama hakkında bir pamuk fide. <img src="/files/ftp_upload/2938/2938fig2.jpg" alt="Şekil 2" /> Şekil 2 Agrobacterial geçişlerini kolaylaştırmak için bir 25 G iğne kullanarak pamuk bitkileri alt kotiledonlarında üzerinde küçük delikler Delme. <img src="/files/ftp_upload/2938/2938fig3.jpg" alt="Şekil 3" /> Şekil 3 yaralama siteleri aracılığıyla pamuk kotiledonlarında içine Agrobacterial karışımı El infiltrasyon iğnesiz bir şırınga kullanarak <img src="/files/ftp_upload/2938/2938fig4.jpg" alt="Şekil 4" /> Şekil 4 Albino fenotip pamuk bitkileri susturuldu VIGS çıktı. Dört çeşitlerin gösterilmiştir. A) Fibermax 832 B) Phytogen 480WR C) Phytogen 425RF D) Deltapine 90.

<ol>
	<li>Chen, Z. J., Scheffler, B. E., Dennis, E., Triplett, B. A., Zhang, T., Guo, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+sequencing+cotton+(Gossypium)+genomes.">Toward sequencing cotton (Gossypium) genomes.</a> Plant Physiol. 145, 1303-1310 (2007).</li><li>Dinesh-Kumar, S. P., Anandalakshmi, R., Marathe, R., Schiff, M., Liu, Y. <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.">Virus-induced gene silencing.</a> Methods Mol Biol. 236, 287-294 (2003).</li><li>Hamilton, A. J., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+species+of+small+antisense+RNA+in+posttranscriptional+gene+silencing+in+plants.">A species of small antisense RNA in posttranscriptional gene silencing in plants.</a> Science. 286, 950-952 (1999).</li><li>Burch-Smith, T. M., Anderson, J. C., Martin, G. B., 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=Applications+and+advantages+of+virus-induced+gene+silencing+for+gene+function+studies+in+plants.">Applications and advantages of virus-induced gene silencing for gene function studies in plants.</a> Plant J. 39, 734-746 (2004).</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 J. 31, 777-786 (2002).</li><li>Estevez, J. M., Cantero, A., Romero, C., Kawaide, H., Jimenez, L. F., Kuzuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+expression+of+CLA1,+a+gene+that+encodes+the+1-deoxyxylulose+5-phosphate+synthase+of+the+2-C-methyl-D-erythritol-4-phosphate+pathway+in+Arabidopsis.">Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis.</a> Plant Physiol. 124, 95-104 (2000).</li><li>Mandel, M. A., Feldmann, K. A., Herrera-Estrella, L., Rocha-Sosa, M., Leon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLA1,+a+novel+gene+required+for+chloroplast+development,+is+highly+conserved+in+evolution.">CLA1, a novel gene required for chloroplast development, is highly conserved in evolution.</a> Plant J. 9, 649-658 (1996).</li><li>Gao, X., Wheeler, T., Li, Z., Kenerley, C., He, P., Shan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+GhNDR1+and+GhMKK2+compromised+cotton+resistance+to+Verticillium+wilt.">Silencing GhNDR1 and GhMKK2 compromised cotton resistance to Verticillium wilt.</a> Plant J. 66, 293-305 (2011).</li></ol>

Çıkar çatışması ilan etti.

VIGS, endojen genlerin ifade geçici olarak aşağı vurma işlevsel genomik analiz güçlü bir araç olduğu kanıtlanmıştır. Bu çalışmada, biz TRV tabanlı bir ikili vektör kullanan bir Agrobacterium VIGS geliştirdi. Pamuk CLA1 (GrCLA1) gen susturma etkinliğini izlemek için görsel bir belirteci olarak geliştirilmiştir. Biz sürekli gen susturulması verimliliği% 100, yaklaşık iki hafta yazılan infiltrasyon başlayarak, test tüm çeşitleri gerçek yaprakları üzerinde görülen albino feno...

<table border="1"><tbody><tr><th> Reaktifler ve ekipmanları Adı </th><th> Şirket </th><th> Katalog / seri numarası </th><th> Yorumlar </th></tr><tr><td> Rulo davul </td><td> Glas-Col, LLC. </td><td> 099A TC108 </td><td> Agro-bakteri kültürü </td></tr><tr><td> Kuvöz </td><td> Sheldon Üretim, Inc. </td><td> 01046209 </td><td> Agro-bakteri kültürü </td></tr><tr><td> UV / VIS Spektrofotometre </td><td> Beckman Coulter </td><td> Model: DU530 </td><td> Ölçüm OD </td></tr><tr><td> Gene Verici </td><td> BioRad </td><td> Model: 1652076; Seri: 154BR3880 </td><td> Elektroporasyon </td></tr><tr><td> Verici Kontrolörü </td><td> BioRad </td><td> Model: 1652098; Seri: 232BR4833 </td><td> Elektroporasyon </td></tr><tr><td> Micropulser Elektroporasyon küvet </td><td> BioRad </td><td> 165-2081 </td><td> Elektroporasyon </td></tr><tr><td> 1 ml Şırınga </td><td> BD Biosciences </td><td> 30962 </td><td> Agro-bakteri, Aşılama </td></tr><tr><td> Metro Mix 700 </td><td> SUNGR </td><td> SKU # 553.001 </td><td> Büyüyen fide </td></tr><tr><td> Terra Cotta pot </td><td> TOPlastics </td><td> GPS 3001B2 </td><td> Büyüyen fide </td></tr><tr><td> MES monohidrat </td><td> USB </td><td> 18886 </td><td> Sızma tampon </td></tr><tr><td> Acetosyringone </td><td> Sigma </td><td> D134406 </td><td> Sızma tampon </td></tr></tbody></table>

agrobacterium-mediated virus-induced gene silencing assay in cotton

Pamuk (Gossypium hirsutum), dünya çapında en önemli bitkileri biridir . , Yeni çeşitlerin moleküler ıslahı konusunda önemli çabalar yapılmıştır. Genomu, gen çoğaltılması ve poliploidi, genetik transformasyon 1 uzun büyüme döngüsü ve inatçılıklarıyla büyük boyutu nedeniyle büyük olasılıkla modern bitki türleri, en gerisinde kalmıştır pamuk büyük ölçekli gen fonksiyonel analiz edilmiştir . Yüksek pamuk üretilen fonksiyonel genetik / genomik çalışma kolaylaştırmak için, pamuk gen fonksiyonlarını değerlendirmek için hızlı ve etkili bir geçici testleri geliştirmek için çalışırlar. Virüs Bağlı Gen susturma (VIGS), viral çoğalma 2,3 bastırmak için ana Post-transkripsiyonel gen susturma (PTGS) dayalı olarak geliştirilen güçlü bir tekniktir. Agrobacterium VIGS Solanaceae, Arabidopsis ve baklagil türlerinin ikiçeneklilerin türlerin geniş bir yelpazede başarıyla uygulanan ve biçeneklilerin türler arpa, buğday ve mısır, çeşitli fonksiyonel genomik çalışmalar için 3,4 dahil olmuştur. Bu hızlı ve verimli bir yaklaşım bitki dönüşüm önler ve fonksiyonel fazlalık üstesinden, özellikle çekici ve dönüşüm için uygun değildir pamuk gibi bitki türleri işlevsel genomik çalışmaları için uygundur. Bu çalışmada, pamuk Agrobacterium VIGS sisteminin ayrıntılı protokol rapor. Çeşitli viral VIGS vektörleri arasında, tütün çıngırak virüsü (TRV) ev sahipliği yapan geniş bir yelpazede işgal etti ve henüz hosts5 hafif belirtiler üretmek tüm bitki boyunca kuvvetlice yaymak mümkün. Susturulması verimliliği, GrCLA1 ​​alterados 1 geni (AtCLA1) pamuk, CLA1 gen kloroplast gelişimi 6 yer almaktadır klonlanmış ve VIGS ikili vektör pYL156 takılı olan ve önceki çalışmalar göstermiştir Arabidopsis Cloroplastos bir homolog gen izlemek için AtCLA1,-fonksiyon kaybı, verimlilik susturmak için mükemmel bir görsel belirteç sağlayarak, gerçek yaprakları 7 bir albino fenotip sonuçlandı . Yaklaşık iki haftada sonrası Agrobacterium infiltrasyon, albino fenotip çoğaltılan tüm deneylerde% 100 susturulması verimliliği ile, gerçek yaprakları görünmeye başladı. Susturulması endojen gen ekspresyonu RT-PCR analizleri ile de teyit edilmiştir. Önemli ölçüde, susturulması kuvvetli Texas çeşitli ticari olarak yetiştirilen çeşitler de dahil olmak üzere, test edilen tüm çeşitlerin meydana gelebilir. Bu hızlı ve verimli Agrobacterium VIGS tahlil pamuk genom düzeyinde gen fonksiyonları hızlı bir şekilde büyük çaplı bir analiz için çok güçlü bir araç sağlar.

Watch this Scientific Journal Video about Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton at JoVE.com

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Agrobacterium pamuk virüs indüklenen gen susturulması (VIGS) tahlil için ayrıntılı protokol sunuyoruz. Tütün çıngırak virüsü (TRV) türetilen VIGS vektörler, pamuk GrCLA1, Cloroplastos alterados 1 geninin RNA susturma ikna etmek için konuşlandırıldı. Susturulması GrCLA1 ​​kaynaklanan albino fenotip fide aşamasında aşılama sonrası 2 hafta içinde gözlenmiştir.

Pamuk olarak Agrobacterium Virüs Bağlı Gen susturma Testi

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

agrobacterium-mediated-virus-induced-gene-silencing-assay-in-cotton

Research

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Biology

24.3K Görüntüleme.  Texas A&M University. Agrobacterium pamuk virüs indüklenen gen susturulması (VIGS) tahlil için ayrıntılı protokol sunuyoruz. Tütün çıngırak virüsü (TRV) türetilen VIGS vektörler, pamuk GrCLA1, Cloroplastos alterados 1 geninin RNA susturma ikna etmek için konuşlandırıldı. Susturulması GrCLA1 ​​kaynaklanan albino fenotip fide aşamasında aşılama sonrası 2 hafta içinde gözlenmiştir.

Video: Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

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.

Virüs indüklenen gen susturma (VIGS) Nicotiana benthamiana Ve Domates

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

Biyoloji

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.

Canlı Hücre Kalsiyum Görüntüleme siRNA aracılığı ile gen susturma ile birlikte Ca tanımlar 2 + ER Membran Kaçak Kanallar ve Düzenleme Mekanizmaları

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

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.

Hücre Kültürü ve Hipoksi Test İndüksiyon

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 ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi-aracılı Gene Nakavt ve<em&gt; In Vivo</emYetişkin Kadınlar&gt; diürez Testi<em&gt; Aedes aegypti</em&gt; Sivrisinekler

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

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.

Ribozom Bağlı Doğan Polipeptitler İzolasyonu<em&gt; In vitro</em&gt; MRNA boyunca Translasyonel Duraklat Siteleri Belirleme

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.

Ribozom Ciltli Polipeptitler izole bir aracı olarak SECM Tutuklanma sırası kullanılarak

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

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.

Optimizasyon ve Kullanımı<em&gt; Agrobacterium</emIn&gt; aracılı Geçici Protein Üretimi<em&gt; Nicotiana</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.

Hastalık Vektör Sivrisinek Larva içinde Kitosan / Müdahale RNA Nanopartikül Aracılı gen susturma

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.

SiRNA aracılı susturma Mouse Oositlerinin içinde mayoz Mili Değerlendirmesi

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

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

İki adımlı PCR ve Yeni Nesil 16S rRNA Gen Sıralaması Kullanarak Mikrobiyota Analizi

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...