İş hibe no tarafından desteklenmiştir. &quot;Bundesministerium für Forschung und Technologie (BMBF) 01GU0615.

1) shRNAs Bakteriyel Teslimat <ol><li> Bakteri kültürü ile başlamadan önce, bir LB-orta ve LB-agar hazırlamak zorundadır. </li><li> LB-orta maya özü (% 0.5 w / v), bacto Tripton (% 1.0 w / v) ve NaCl (% 0.6 w / v), tartmak ve aqua bidest bu bileşenleri sulandırmak için. Hazırlanan çözümler otoklavda sterilize olması ve sonra kullanıma hazır. </li><li> LB-agar plaklarına için bacto agar (% 1.5 w / v) sterilizasyon önceki LB-orta eklenecek. </li><li> LB-agar, mikrodalga, LB-agar tamamen eriyene kadar isitin. Şişe yandı almadan kolayca dokundu kadar çözüm soğumaya alalım. Daha ileri adımlar olumlu klonların seçimi için kanamisin (100 mg / ml) ekleyin. </li><li> LB-agar plakları 10 cm Petri kaplarına 20 ml LB-agar çözüm pipetleme laminer akış tezgah (steril koşullar) hazırlayın. Plakalar, sıvı katı hale gelene kadar soğumaya alalım. Plakalar, artık kullanıma hazır ve 4 ° C&#39;de saklanabilir. </li><li> ShRNA-kodlaması ekspresyon vektörleri yetkili E. dönüştü. CaCl 2 yordamı kullanarak ısı şok coli ceq221. </li><li> Plaka 100 mcg / ml kanamisin içeren LB-agar plaklarına dönüştürülmüş bakteri, kapağını kapatın parafilm ile plakaları ve baş aşağı 37 ° yetiştirmek ° C, gece boyunca. </li><li> Diş çekme ile yetiştirilen koloniler seçerek olumlu klonları ile bakteriyel mini kültürleri inoküle edin. Diş, 100 mg / ml kanamisin ve ° C gece boyunca 200 rpm&#39;de çalkalayıcı üzerinde 37 yetiştirmek içeren taze LB-orta 7,0 ml içine almak aktarın. </li><li> İnokülasyon 100 ml taze LB-orta, 37 ° C gece boyunca 200 rpm&#39;de çalkalamalı üzerinde gece 1 litre Erlenmeyer şişesinin içinde bir mini kültür ve inkübe ile 100 mg / ml kanamisin 1:100 içeren </li><li> Tohum 25 x 10 4 6 başına insan mide kanseri hücreleri (EPG85-257RDB) (numaralı seribaşı hücrelerinin sayısına göre hücre hattı hücre büyüme hızına bağlı confluency ~ tohumlama 24 saat sonra% 70-80 olmalıdır) iyi yemekler ve bu inkübe 37 ° C,% 5 CO 2, gece boyunca su buharına doymuş atmosfer. </li><li> Kanser hücresi enfeksiyon Önceki gece tk RNAi vektör içeren E. kültürler üzerinde coli OD 600 belirlemek için bir fotometre ölçülür. 0.5 optik yoğunluk kadar bakteri çözeltinin (OD 600 = 0.5, 1.6 x 10 8 bakteri hücre / ml eşittir) ulaştı . </li><li> FCS-co-inkübasyon bakteriyel önceki 30 dakika orta karşı karsinom hücrelerinin FCS içeren hücre kültür ortamı değiştirin. </li><li> 1 x PBS ile iki kez bakteri yıkayın ve serum serbest Leibovitz L-15 orta sulandırmak. </li><li> Istenen İçişleri Bakanlığı 37 kanser hücreleri (bakteri hücrelerinin kanser hücre başına enfeksiyon çokluğu = sayı) ve co-inkübe bakteri ve 2 saat boyunca kanser hücreleri için seyreltilmiş bakteri ° C </li><li> Co-inkübasyon kanser hücrelerinin 2 saat 100 u / ml penisilin, 100 mcg / ml streptomisin, 2.5 mikrogram / ml amfoterisin, 150 mikrogram / ile desteklenmiş serum içeren Leibovitz L-15 orta bir kez 1x x PBS ile iki kez yıkanır ve sonra ml gentamisin ve 100 mg / ml kanamisin. </li><li> 37 kanser hücrelerinin yetiştirilmesi ° C,% 5 CO 2, su buharına doymuş atmosfer devam edin. </li><li> MRNA düzeyinde kantitatif gerçek zamanlı RT-PCR ile ölçülen floresan mikroskobu, bakteriyel tedavi etkileri ve protein düzeyi western blot analizi ile görüntülenebilmekte ve FACS analiz ve hücre proliferasyon deneyleri gibi fonksiyonel testleri ile gösterilir. (Isteniyorsa, bu işlemler ayrıntılı olarak tarif edilebilir). </li></ol> 2) Temsilcisi Sonuçlar Protokolü doğru yapılırsa, sonuçlar aşağıda gösterilen olanlar ile karşılaştırılabilir olmalıdır. Floresan mikroskopi Şekil 1 bakteriyel üç saat sonra bir insan mide kanseri hücre karşılaştırıldığında işlenmemiş bir hücre (b) (a) gösterir. Tedavi edilen hücre çekirdeğinin etrafında, bakteri tespit edilebilir. <img src="/files/ftp_upload/2099/2099fig1.jpg" alt="Şekil 1" /> Şekil 1: Floresan mikroskobu (1:500) sonra bakteriyel İnsan mide kanseri hücre ve işlenmemiş bir insan mide kanseri hücre kontrolü, DAPI boyama, DAPI bandpass (Λem = 640 nm), 40x objektif. Kantitatif gerçek zamanlı RT-PCR Rakam 2 yaklaşık% 70 MDR1 mRNA down-regülasyonu (siyah ışın) tedavi bakteri ile tedavi sonrası görülebilir. Ebeveyn hücreleri MDR1 aşırı ekspresyonu eksikliği nedeniyle pozitif kontrol olarak hizmet vermektedir. 257RDB P170 içeren hücre hattı plazmid bir anti-MDR1 shRNAs s olarak alınan diğer R transkingdom RNAi teknolojisi doğrudan karşılaştırma ifadeNAI stratejileri susturmak. Pozitif kontrol olarak tedavi edilmezse dayanıklı hücre hattı EPG85-257RDB eksprese MDR1, plazmid ifade shRNA eksik bakteri ile tedavi aynı hücre hattı ve bu hücre hattı taşıyan bir plazmid ifade anti-MRP2 shRNAs tedavi bakteri ile tedavi alındı. <img src="/files/ftp_upload/2099/2099fig2.jpg" alt="Şekil 2" /> Şekil 2: terapötik E. ile tedavi sonrası kantitatif gerçek zamanlı RT PCR MDR1 mRNA anti-MDR1 shRNAs (500 İçişleri Bakanlığı 1) ifade coli ceq221. Normalizasyon housekeeping gen aldolase kullanılarak yapıldı. Tedavi edilmezse hücrelerin hücre hattı EPG85-257RDB MDR1/aldolase oranı% 100 olarak belirlenmiştir. P-değerleri öğrencinin t-testi kullanılarak hesaplandı (* = p &lt;0.05, ** p = &lt;0.005, *** p &lt;0,001). Western Blot analizi Şekil 3 MDR1 aşağı yönetmelik bakteriyel tedavi sonrasında protein düzeyi de yer aldığını ortaya koyuyor. Bu ilaç duyarlı ebeveyn hücre hattı (EPG85-257P), tedavi edilmezse ilaca dirençli hücre hattı (EPG85 257RDB) MDR1 ifade ve tedavi örnek MDR1 ifade eksik MDR1 ifade gösterir. Bakteriyel tedavi hücrelerinin MDR1 down-regülasyonu, açık bir şekilde görülebilir. <img src="/files/ftp_upload/2099/2099fig3.jpg" alt="Şekil 3" /> Şekil 3: Western blot analizi MDR1 ekspresyon düzeyleri tedavi edilmezse uyuşturucu duyarlı EPG85-257P, E. ile birlikte inkübasyondan sonra tedavi edilmezse ilaca dirençli EPG 257RDB ve EPG85-257RDB. coli ceq221 + P43 MDR1. İlköğretim Ab C219 1:100 ve anti-aktin 01:05 000, ikincil Ab anti-fare 1:10 000. Sitotoksisite tahlil Sitotoksisite testinde şekil 4&#39;deki gibi Fonksiyonel analiz transkingdom RNAi işleyişi için de göstergeleridir. Ebeveyn hücre hattı ve Anıt-MDR1 shRNA plazmid ifade içeren hücre hattı daunorubisin için herhangi bir direnç görünmüyor. Dayanıklı hücre hattı EPG85-257RDB ve iki tane daha kontrol daunorubisin için yaklaşık% 90 oranında direnç tersine anti-MDR1 shRNA ifade bakteri ile tedavi örnek göre direnç önemli bir değişiklik görünmüyor. <img src="/files/ftp_upload/2099/2099fig4.jpg" alt="Şekil 4" /> Şekil 4: Sitotoksisite tahlil. İlaç-özel hücrenin hayatta kalabilmek için bir sitotoksisite testinde tarafından belirlenir IC50 değerleri. P-değerleri öğrencinin t-testi kullanılarak hesaplandı (* = p &lt;0.05, ** p = &lt;0.005, *** p &lt;0,001). Antrasiklin birikimi tahlil Şekil 5 de gösterildiği gibi bakteriyel tedavi numunelerin mukavemet azaldığı (Şekil 4) göre, anti-MDR1 shRNA muamele edilen hücreler antrasiklin birikiminin yaklaşık% 90 oranında artmıştır. Ebeveyn hücre hattı ve hücre hattı 257RDB P170 güçlü bir daunorubisin birikimi% 100 gösteriyor. Dayanıklı varyant nadiren herhangi bir birikimi gösterir. Anti-MDR1 shRNA ifade bakteri ile tedavi edilen hücreler,% 90 bir anthracyline birikimi artış göstermektedir. <img src="/files/ftp_upload/2099/2099fig5.jpg" alt="Şekil 5" /> Şekil 5: Antrasiklin birikimi tahlil. Antrasiklin flow sitometri ile ölçülen bakteriyel 6 gün sonra karsinom hücrelerinin birikimi. P-değerleri öğrencinin t-testi kullanılarak hesaplandı (* = p &lt;0.05, ** p = &lt;0.005, *** p &lt;0,001).

<ol>
	<li>Kr&#252;hn, A., Wang, A., Fruehauf, J. H., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+short+hairpin+RNAs+by+transkingdom+RNA+interference+modulates+the+classical+ABCB1-mediated+multidrug-resistant+phenotype+of+cancer+cells.">Delivery of short hairpin RNAs by transkingdom RNA interference modulates the classical ABCB1-mediated multidrug-resistant phenotype of cancer cells.</a> Cell Cycle. 8, 3349-3354 (2009).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDR1/P-glycoprotein+(ABCB1)+as+target+for+RNA+interference-mediated+reversal+of+multidrug+resistance.">MDR1/P-glycoprotein (ABCB1) as target for RNA interference-mediated reversal of multidrug resistance.</a> Curr. Drug Targets. 7, 813-821 (2006).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+cancer+multidrug+resistance:+a+still+unsolved+problem.">An overview of cancer multidrug resistance: a still unsolved problem.</a> Cell. Mol. Life Sci. 65, 3145-3167 (2008).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+RNA+interference+in+drug-resistant+cancers.">Therapeutic potential of RNA interference in drug-resistant cancers.</a> Fut. Oncol. 5, 169-185 (2009).</li><li>Nguyen, T. A., Fruehauf, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transkingdom+RNA+interference+(tkRNAi):+a+novel+method+to+induce+therapeutic+gene+silencing.">Transkingdom RNA interference (tkRNAi): a novel method to induce therapeutic gene silencing.</a> Methods Mol. Biol. 514, 27-34 (2009).</li><li>Nieth, C., Priebsch, A., Stege, A., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+classical+multidrug+resistance+(MDR)+phenotype+by+RNA+interference+(RNAi.">Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi.</a> FEBS Lett. 545, 144-150 (2003).</li><li>Stege, A., Priebsch, A., Nieth, C., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+and+complete+overcoming+of+MDR1/P-glycoprotein-mediated+multidrug+resistance+in+human+gastric+carcinoma+cells+by+RNA+interference.">Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference.</a> Cancer Gene Ther. 11, 699-706 (2004).</li><li>Stein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+in+vivo+reversal+of+the+multidrug+resistance+(MDR)+phenotype+by+jet-injection+of+anti-MDR1+short+hairpin+RNA-encoding+plasmid+DNA.">Complete in vivo reversal of the multidrug resistance (MDR) phenotype by jet-injection of anti-MDR1 short hairpin RNA-encoding plasmid DNA.</a> Mol. Ther. 16, 178-186 (2008).</li><li>Xiang, S., Fruehauf, J., Li, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-expressing+bacteria+elicit+RNA+interference+in+mammals.">Short hairpin RNA-expressing bacteria elicit RNA interference in mammals.</a> Nat. Biotechnol. 24, 697-702 (2006).</li></ol>

Hücre sayısı enfeksiyonu numaralı seribaşı ve ilgili İçişleri Bakanlığı, kritik hücre hatları ve gözlem altında büyüme hızı bağlıdır. Tohumlama için en uygun hücre sayıları bulmak için, ön deneyler büyüme hızını belirlemek için önemle tavsiye edilir. Bunun yanı sıra, farklı Mois hücreleri stres ölmeden bakteri istilası durabiliriz hangi uzatmak nedeniyle sınırlı test edilmelidir. Gözlem altında gen aşağı düzenleme değişebilir zaman en iyi noktası. En uygun koşullar?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>214050</td>
<td>&nbsp;</td>
<tr>
<td>Amphotericin B</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2612</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline-PBS (10 x)</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>14080048</td>
<td>&nbsp;</td>
<tr>
<td>E. coli ceq221</td>
<td>&nbsp;</td>
<td>Cequent Pharmaceuticals Inc.</td>
<td>&nbsp;</td>
<td>No catalogue available, direct order</td>
</tr>
<tr>
<td>Gentamycin</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2710</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>&nbsp;</td>
<td>Contains 5,000 units of penicillin (base) and 5,000 &mu;g of streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline.</td>
</tr>
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>Merck KgaA</td>
<td>1064060500</td>
<td>&nbsp;</td>
<tr>
<td>Tryptone</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>211705</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>212750</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

bacterial delivery of rnai effectors: transkingdom rnai

RNA enterferans (RNAi) mRNA spesifik inhibisyonu için yüksek etkili bir mekanizma temsil eder. Yanı sıra, güçlü bir laboratuar aracı olarak potansiyel RNAi yolu terapötik kullanımı için umut verici görünüyor. RNAi RNA girişim (RNAi) tabanlı tedaviler, teslim geliştirilmesi için aracılık hedef hücreleri ajanların en büyük engellerden biridir. Bu engeli aşmak için yeni bir strateji transkingdom RNAi (Tk RNAi). Bu teknoloji üretmek ve tedavi kısa saç tokası RNA (shRNA) teslim RNAi neden hedef hücrelerine, patojen olmayan bakteriler, örneğin Escherichia coli kullanır. İlk nesil tk RNAi-aracılık vektörü, TURU, terapötik bir ilgi shRNA ifade düzenlenmesi için bakteriyofaj T7 organizatörü içerir. Ayrıca, TRIP doğal noninvaziv bakteri β1-integrin pozitif memeli listeriolysin O. Bu enzim üreten hücreleri ve Listeria monocytogenes HlyA gen, girmek için izin verir invasin kodlar Yersinia pseudotuberculosis Yat odağı, tedavi shRNA kaçmak sağlar giriş, hedef hücre sitoplazma içinde vezikül. TRIP yapıları shRNAs, T7 organizatörü odaklı sentezi için gerekli olan T7 RNA polimeraz kodlar yetkili bir patojen olmayan Escherichia coli suşu, içine tanıtıldı . Farklı RNAi stratejileri için iyi karakterize bir kanser ilişkili hedef molekülü ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). Bu bir ilaç ekstrüzyon pompa ve aracılık &quot;klasik&quot; ABCB1-aracılı çoklu ilaç direnci (MDR) belirli bir çapraz direnç paterni ile karakterize olan insan kanser hücrelerinin fenotipi olarak ABC taşıyıcı davranır. Farklı ABCB1 ifade MDR kanser hücrelerinin anti-ABCB1 shRNA ifade vektör taşıyan E. ile tedavi edildi coli. Bu prosedür içinde RNAi yollarının kanser hücrelerinin aktivasyonu ve ABCB1 kodlama mRNA önemli bir down-regülasyonu yanı sıra ilgili ilaç ekstrüzyon pompa sonuçlandı. Buna göre, ilaç birikimi bozulmamış ilaca dirençli kanser hücrelerinin gelişmiş ve MDR fenotip tersine dönmüştür. Bu model sayesinde veri tk RNAi insan kanser hücreleri kanser ilişkili faktörler, örneğin ABCB1 modülasyonu için uygun olduğunu proof-of-concept sağlar.

Watch this Scientific Journal Video about Bacterial Delivery of RNAi Effectors: Transkingdom RNAi at JoVE.com

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA enterferans (RNAi) tabanlı tedaviler geliştirilmesi için, yeni bir strateji, transkingdom RNAi (tkRNAi) geliştirildi. Bu teknoloji üretmek ve hedef hücrelerine terapötik kısa saç tokası RNA (shRNA) teslim etmek için non-patojenik bakterilerin kullanır. Burada, tkRNAi klasik kanser hücrelerinin ABCB1 aracılı çoklu ilaç direnci (MDR) iptali için başarıyla uygulanmıştır.

RNAi Efektör Bakteriyel Teslim Tarihi: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

bacterial-delivery-of-rnai-effectors-transkingdom-rnai

Research

JoVE Journal

Biology

13.4K Görüntüleme.  Charité Campus Mitte. RNA enterferans (RNAi) tabanlı tedaviler geliştirilmesi için, yeni bir strateji, transkingdom RNAi (tkRNAi) geliştirildi. Bu teknoloji üretmek ve hedef hücrelerine terapötik kısa saç tokası RNA (shRNA) teslim etmek için non-patojenik bakterilerin kullanır. Burada, tkRNAi klasik kanser hücrelerinin ABCB1 aracılı çoklu ilaç direnci (MDR) iptali için başarıyla uygulanmıştır.

Video: Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Yetişkin Sivrisinekler (A. gambiae) RNAi Tahliller için protokol

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Biyoloji

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

Lokalize RNAi ve Tıbbi Sülük Ektopik Gen İfadesi

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

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

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

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

Büyüme inhibisyonu fenotipleri tanımlanması Mayalarda bakteriyel Tip III Efektör İfade İndüklenmiş

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

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

Memeli Hücreleri RNAi Tarama MİSYON esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

DsRNA Enjeksiyon RNAi Girişim içine Drosophila Embriyolar

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAi Necromenic nematodu Mikroenjeksiyon Gen demonte ve Transgenesis aracılığı ile Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

Içinde Postembryonic Fenotiplerin Belirleme RNAi Tarama C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Nicel ve Otomatik Yüksek throughput Genom çapında RNAi Ekranlar C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

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 ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

Kemirgen Epididimal Spermatozoa&#39;lar phosphopeptide Analizi

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...