Biz laboratuarımızda üyeleri, verimli tartışmalar ve teknik yardım için teşekkür ederim. Bu video sunum Araştırma ve Veteriner patobiyolojisi Anabilim Dalı Dekan Yardımcısı, Veteriner Sağlık Bilimleri Merkezi, Oklahoma State University tarafından desteklenmiştir. Araştırma Ministerio de Ciencia e Innovación, İspanya (proje BFU2008-01244/BMC), JF CSIC intramural proje PA1002451 tarafından finanse edildi, Walter R. Sitlington KMK, CVHS 2009 RAC hibe, OAES Gıda Hayvancılık Araştırma Başkanı donatılmış Hayvan Sağlığı Fonları ve USDA, Ulusal Araştırma Girişimi Rekabetçi Grant, No 2.007-04.613.

1. DsRNA, Üretimi. <ol><li> DsRNA in vitro transkripsiyon ve sentez (örneğin, Dermacentor subolesin oligonükleotid primerler D8AAT75 variabilis için T7 promotor dizisinde içeren oligonükleotid primerler sentez: 5&#39;-TAATACGACTCACTATAGGGTACTGACTGGGATCCCCTGCACAGT-3 &#39;ve D8DVT73: 5&#39;-TAATACGACTCACTATAGGGTACTCGAGCTTGGTGGAAAGGACG-3&#39;). </li><li> RT-PCR her oligonükleotid astar ve kene toplam RNA 1-10 ng 10 pmol kullanarak hedef gen artırın. </li><li> PCR ürünü arındırın. </li><li> DsRNA 8 mcL saflaştırılmış PCR ürünü kullanarak sentezlerler. </li><li> Spektrometresi ile dsRNA sayısal olarak. </li></ol> 2. DsRNA ile Keneler enjeksiyon. 2.1. Keneler enjeksiyon için hazırlanması. <ol><li> Öncelikle, her çözüm, tek bir 50 ml santrifüj tüpüne onları sallayarak tüp üst keneler korumak için üzerine ince bir örgü tel ekran yoluyla çözüm şişeden çözümleri bir dizi keneler yıkayın. Keneler çamaşır için çözümler dizisi, musluk suyu,% 3 hidrojen peroksit, distile su,% 70 etanol ve saf su ile iki kez daha yıkar iki yıkar. </li><li> Kağıt havlu üzerinde kene Blot kurulayın. </li><li> Deney bağlı olarak 20 ila 50 arasında gruba keneler Sayısı 1.25 oz plastik bardak, deneysel grup numarası ile sıkı donatılmış bir kapak ve etiket ile her gruptan keneler. </li></ol> 2.2. Enjeksiyon ekibi işaretleyin. RNAi ekip üç kişiden oluşur: (1) kırmızı diş balmumu bir sayfasına yapıştırılmış çift yapışkan bant, her kene pozisyonları bir kişi, (2), sonra kenelerin izler ve keneler (3) bir kişi enjekte bir kişi enjeksiyon, keneler bunları etkinleştirmek için CO 2 nefes ve deneysel grup numarası ile etiketlenmiş bardak içine yaşayan keneler sayar. Tüm ekip üyeleri, tek kullanımlık eldivenler giymelidir. 2.3. Enjeksiyon için keneler yerleştirilmesi. <ol><li> Dumont ince forseps kullanarak kene yakalayın ve kırmızı diş balmumu 3 &quot;x 6&quot; levha yapıştırılmış çift yapışkan bant üzerinde ventral tarafında bir yer. Keneler 5 keneler gruplar halinde bir araya yakından konumlandırılmış. </li><li> Tüm 5 kenelerin ağız onları daha da dizginlemek için küçük bir şerit üzerinde maskeleme bandı yerleştirin ama vücudun en enjeksiyon işlemi, kene enjektör (Şekil 1) tarafından görülebilir bu yüzden maruz bırakarak. </li></ol> 2.4. Keneler Enjeksiyon. <ol><li> Keneler Exoskeleton ventral yüzeyinde sağ alt kadranda enjekte edilecektir. </li><li> İlk olarak, dış iskelet, ½ &quot;, 29 gauge iğne (Şekil 2a) ile donatılmış bir Monoject insülin şırınga kullanarak bir delik delmek. </li><li> 45 ° Eğimli noktası ile 1 inç, 33 gauge iğne ile ısmarlama Hamilton şırınga kullanarak (mcL başına 5 x 10 11 moleküller 5 x 10 10) (Şekil 2b) dsRNA çözüm 0.2-0.5 mcL hemen kene enjekte . Kene boşluğuna iğne dsRNA yerleştirilmesi ve saklama sigortalamak için iyi yerleştirilmiş olmalıdır. Bazı sıvı enjeksiyon yerinde (Şekil 2c) kaçmak için muhtemeldir. Üzerinde hemolimf kaybına neden olan keneler, enjekte etmek ve kene ölüme neden olabilir alınmalıdır. </li><li> Hamilton şırınga her deney grubunda enjeksiyonları tamamladıktan sonra temizleyin. başka bir deney grubu için kullanmadan önce. Bir atık kabı içine kovmak ve 15 kez tekrarlayın sonra beher% 3 hidrojen peroksit içeren bir şırınga doldurun ve. Steril su içeren bir beher şırınga doldurun ve sonra bir atık kabı içine kovmak ve 15 kez tekrarlayın. Bükülmüş ise, piston düzgün hareket ve nazik bir dokunuş keneler enjeksiyon için gerekli cevap, çünkü Hamilton şırınga pistonu eğmemeye dikkat edin. </li></ol> 2.5. Enjeksiyondan sonra kene tedavisi. <ol><li> Ince forseps ile hemen çift yapışkan bant enjekte kene Pick up ve plastik bir kurtarma konteyner (yaklaşık 6 &quot;x 6&quot; ve keneler kaçmasını önlemek için maskeleme bandı ile halkalı) yerleştirin. Keneler, enjeksiyondan sonra kısa bir süre devre dışı olacak ama yakında çanak etrafında tarama başlamalıdır. </li><li> Nefes kurtarma konteyner keneler etkinleştirilmesine yardımcı olmak için onları yerleştirerek hemen sonra kenelerin üzerine 2 CO. Sonra, tarama ve keneler aktif enjeksiyon yara hızla iyileşir ve bunlar büyük olasılıkla hayatta kalacaktır. </li><li> Her deney grubu sayısına göre keneler sayın ve sıkıca monte kapaklı etiketli bir plastik bardak koyun. Yedek keneler herhangi bir sonraki deney grubu enjekte önce öldüğünü herhangi bir yerine enjekte edilmelidir. </li></ol> 2.6. Tutarak işaretleyin. <ol><li> Nem odasında keneler (12saat ışık Yeri: 22-25 az 12 saat karanlık fotoperiyodun ° C ve% 95 bağıl humidity) ve 1 gün için basılı tutun. </li><li> Yeri keneler, kene besleme hücreleri için bir deney grubu, bir koyun yapıştırılmış ve onları eşit sayıda erkek veya kadın uninjected keneler (enjekte değildi hangisi seks) ile beslenirler. Tıka basa beslemek Kadın keneler, koyun, beslenme 10 gün ya da kontrolü kadın sonra çıkarılır bu konak toplanan ve tartılır düştü. </li><li> Kartonları keneler yerleştirin ve koloni gelişimine tamamlanıncaya kadar nem odasında tutun. Yöntemlerinin koloni ağırlık gruptaki tüm keneler tarafından üretilen yumurta kitle tarafından değerlendirin. </li></ol> 2.7. RNAi sonra kene fenotip analizi. <ol><li> Beslenmeden sonra fenotip kene, keneler, kene kilo yöntemlerinin koloni ve yumurta doğurganlık hayatta sayısını belirleyen değerlendirin. Ancak, çalışmanın hedeflenen gen ve hedeflerine bağlı olarak diğer analizler yapılabilir. </li></ol> 3. RT-PCR ile gen susturma Onayla Analizi. <ol><li> Beslendikten sonra, kontrol enjekte ve dsRNA enjekte grupların bireysel kene tükürük bezleri ve bağırsaklar parçalara ayır. </li><li> Bireysel doku örneklerinden total RNA ayıklayın. </li><li> GenNorm yöntemi (Bio-Rad iQ5 Standart Edition Sürüm 2.0 tarafından uygulanan ddCT) yöntemini kullanarak gerçek zamanlı RT-PCR ile bireysel dokularda hedef gen transkript analiz ve kene 16S karşı RNA düzeyleri normale rRNA. </li><li> Her örnek için aynı sıcaklık aralığında sürekli amplikonlarının doğasını sadece bir Amplikon oluşan olduğundan emin olmak için reaksiyonun sonunda ve disosiasyon eğrileri çalıştırın. </li><li> MRNA düzeyleri kontrol dsRNA enjekte keneler Öğrenci `s, t-testi (P ​​= 0.05) kullanılarak enjekte arasında (normalize Ct değerleri) karşılaştırın. </li></ol> 4. Temsilcisi Sonuçlar: Protokolü burada RNAi için, birçok farklı ixodid kene türlerinde (Tablo 1) laboratuvarda kullanılan olmuştur nitelendirdi. Kene enjekte dsRNA miktarı kene boyutu ile değişir; büyük kene türlerinin daha büyük bir hacim barındırabilir. Negatif kontrol keneler alakasız bir dsRNA ile enjekte edilmelidir. Subolesin 14-19,22-25,27-32,34 ve beta-aktin 20,21 gibi birçok dsRNAs pozitif kontrol olarak kullanılabilir. Karıştırma dsRNA çözümler önlemek için tedaviler arasında şırınga yıkamak için önemli olduğunu unutmayın. Protokolü doğru yapılırsa,% 5 oranında daha az ölüm, 24 saat sonra enjeksiyon prosedürü elde edilmelidir. Keneler gen demonte sonra tipik bir fenotip için ekran koruyucu antijenler kene dsRNA havuzları enjekte keneler bir panel ile Şekil 3 gösterilmiştir. <table border="1"><tbody><tr><td> Türlerin işaretleyin </td><td> dsRNA enjekte </td><td> Referanslar </td></tr><tr><td> Ixodes scapularis </td><td> cDNA kütüphanesi, subolesin, aktin, nükleotidaz, NF-kB, akirin </td><td> 21, 22, 29, 30 </td></tr><tr><td> Dermacentor variabilis </td><td> subolesin, GST, ubikuitin, vATPase, selenoproteinlerin M ve W2A, hematopoietik kök / progenitör hücreleri aktin Proteazom 26S subunit, ferritin1, varisin, akirin, protein gibi </td><td> 15, 19, 22, 24, 26, 30-32 </td></tr><tr><td> Dermacentor marginatus </td><td> subolesin </td><td> 22 </td></tr><tr><td> Amblyomma americanum </td><td> cDNA kütüphanesi, subolesin, akirin </td><td> 17, 22, 30 </td></tr><tr><td> Amblyomma hebraeum </td><td> subolesin, voraxin </td><td> 28 </td></tr><tr><td> Rhipicephalus sanguineus </td><td> Rs86, subolesin </td><td> 22, 23 </td></tr><tr><td> Rhipicephalus Microplus </td><td> GST, ubikuitin, selenoprotein, Bm86, Bm91, subolesin, GI, GIII, EF1a flagelliform ipek proteini, von Willebrand faktör </td><td> 16, 18, 25, 27 </td></tr><tr><td> Rhipicephalus annulatus </td><td> ubikuitin, subolesin, EF1a, GIII </td><td> 16 </td></tr></tbody></table> (Tablo 1). RNAi protokolü kullanılan edildiği türlerin işaretleyin. <img src="/files/ftp_upload/2474/2474fig1.jpg" alt="Şekil 1" /> Şekil 1 kırmızı diş balmumu bir sayfasına yapıştırılır çift yapışkan bant keneler Yerleştirme, ventral tarafta. Keneler, küçük bir şerit maskeleme bandı enjektör enjeksiyon sırasında vücut kene gözlemlemek için izin verirken, keneler daha da güvenli amacıyla ağız üzerine yerleştirilir, sonra, 5 gruplar halinde yerleştirilir. <img src="/files/ftp_upload/2474/2474fig2.jpg" alt="Şekil 2" /> Şekil 2 enjeksiyon prosedürü insülin sy kene dış iskelet (a) sağ alt kadranda piercingringe bir enjeksiyon sitesi oluşturmak için 29 gauge iğne ile donatılmış, (b), (c) en olası kene hemolimf bazı kaçak / 33 gauge iğne ile Hamilton şırınga kullanarak bu sitede dsRNA hemen enjeksiyon akışkanlar. <img src="/files/ftp_upload/2474/2474fig3.jpg" alt="Şekil 3" /> Şekil 3 kene altı grup RNAi Amblyomma americanum kene koruyucu antijenler için ekran için kullanılan hangi bir panel. Keneler de fenotipik değişiklikler olumlu subolesin RNAi kontrolü ve olumsuz ilgisiz dsRNA kontrol ile karşılaştırıldığında görülebilir. Bu deneyde, her grubun kene ölüm, ağırlıklar ve yöntemlerinin koloni RNAi etkisi istatistiksel olarak analiz edildi.

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+subolesin+is+an+ortholog+of+the+akirins+described+in+insects+and+vertebrates.">Tick subolesin is an ortholog of the akirins described in insects and vertebrates.</a> Dev. Comp. Immunol. 33, 612-617 (2009).</li><li>Canales, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+and+immunogenicity+of+the+mosquito+ortholog+of+the+tick+protective+antigen,+subolesin.">Conservation and immunogenicity of the mosquito ortholog of the tick protective antigen, subolesin.</a> Parasitol. Res. 105, 97-111 (2009).</li><li>Kocan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+genes+involved+in+Anaplasma+marginale-tick+interactions+affects+the+pathogen+developmental+cycle+in+Dermacentor+variabilis.">Silencing of genes involved in Anaplasma marginale-tick interactions affects the pathogen developmental cycle in Dermacentor variabilis.</a> BMC Dev. Biol. 9, 42-42 (2009).</li><li>Zivkovic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subolesin+expression+in+response+to+pathogen+infection+in+ticks.">Subolesin expression in response to pathogen infection in ticks.</a> BMC Immunol. 11, 7-7 (2010).</li><li>Karim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+tool:+gene+silencing+in+Ixodes+scapularis+eggs+and+nymphs+by+electroporated+dsRNA.">Functional genomics tool: gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA.</a> BMC Biotechnol. 10, 1-1 (2010).</li><li>Kocan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transovarial+silencing+of+the+subolesin+gene+in+three-host+ixodid+tick+species+after+injection+of+replete+females+with+subolesin+dsRNA.">Transovarial silencing of the subolesin gene in three-host ixodid tick species after injection of replete females with subolesin dsRNA.</a> Parasitol. Res. 100, 1411-1415 (2007).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+tick-pathogen+interface+for+novel+control+strategies.">Targeting the tick-pathogen interface for novel control strategies.</a> Front. Biosci. 13, 6947-6956 (2008).</li><li>Kurscheid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+tick+RNAi+pathway+by+comparative+genomics+and+reverse+genetics+screen+of+targets+with+known+loss-of-function+phenotypes+in+Drosophila.">Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila.</a> BMC Mol. Biol. 10, 26-26 (2009).</li></ol>

Diğer yöntemlerle keneler 14, 33 RNAi için tarif olmasına rağmen, dsRNA enjeksiyon beslenmemiş (Tablo 1) ve beslenen keneler 16,25,34 hem de en yaygın kullanılan Burada anlatılan. RNAi kene genin fonksiyonu ile çalışma, kene patojen arayüzü karakterizasyonu ve tarama ve kene koruyucu antijenler 14,35 karakterizasyonu için değerli bir araç olarak görülmüştür. Özellikle, RNAi keneler 35 fonksiyonel analizleri için en değerli bir araç haline gelmiştir. Metodolojik, RNAi muhtemelen bireylerin çok sayıda gen demonte izin verebilir daha verimli yöntemler dönüşecek. Keneler dsRNA bağlı RNAi mekanizması, daha iyi anlaşılmasına ve bu türün 35,36 bu genetik yaklaşım kullanımı katkıda bulunmak için rafine edilmesi gerekir . Keneler de RNAi hedef etkileri ölçüde de tam 14,27 ele olması gereken önemli bir sorudur. Son olarak, RNAi büyük olasılıkla onay gen regülasyonu ve sistem biyolojisi çalışma kapsamlı katkılar sağlayacak ve kene patojen arayüzü ve kene istilası ve iletim kene kaynaklı patojenlerin kontrolü için aşı geliştirilmesi üzerinde bir etkisi olabilir.

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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>Access RT-PCR system</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>A1250</td>
<td>&nbsp;</td>
<tr>
<td>Purelink PCR purification kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K3100-02</td>
<td>&nbsp;</td>
<tr>
<td>Megascript RNAi kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1626M</td>
<td>&nbsp;</td>
<tr>
<td>Red dental wax</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72674</td>
<td>&nbsp;</td>
<tr>
<td>Plastic cups, 1.25 oz and lids</td>
<td>&nbsp;</td>
<td>Solo Cup Company, Urbana Ill.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe</td>
<td>&nbsp;</td>
<td>Monoject</td>
<td>&nbsp;</td>
<td>Fitted with a &frac12;&#x201D;, 29 gauge needle</td>
</tr>
<tr>
<td>Hamilton syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>701SN,33/.375&#x201D;/45DGR</td>
<td>Custom made</td>
</tr>
<tr>
<td>TriReagent</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>93289</td>
<td>&nbsp;</td>
<tr>
<td>iScript One-Step RT-PCR Kit with SYBR Green</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>170-8892</td>
<td>&nbsp;</td>
<tr>
<td>Real-time PCR detection system</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>Several</td>
<td>Please refer to <a href="http://www.bio-rad.com/">http://www.bio-rad.com/</a></td>
</tr>		</tbody>
		</table>
		</div>

rna interference in ticks

Keneler evcil ve vahşi hayvanların ve insanların zorunlu hematophagous ektoparazit ve insan hastalıkları 1 ve en önemli vektörler vektörler sığır sektöründe dünya çapında 2 etkileyen sivrisinekler için dünyada ikinci olarak kabul edilir. Keneler, altsınıf Acari, sipariş Parasitiformes, alttakım Ixodida sınıflandırılır ve Arctic tropikal bölgelerde 3 dünya çapında dağıtılmaktadır . Kene istilası kontrol etmek için tüm çabalarına rağmen, bu ektoparazit 4,5, insan ve hayvan sağlığı için ciddi bir sorun olmaya devam etmektedir. RNA enterferans (RNAi) 6, gen fonksiyonu veya etkisi metabolik yolu belirlemek amacıyla gen ekspresyonu bozulması içeren bir nükleik asit tabanlı ters genetik yaklaşım . Küçük müdahale RNA&#39;lar (siRNA) dsRNA tetik 7-9 gibi aynı sırayla içeren sitoplazmik mRNA&#39;ların güçlü bir dizi özel bozulması, çift iplikli RNA (dsRNA) ve sonuçları tarafından başlatılan RNAi yolun efektör moleküllerdir . DsRNA tarafından başlatılan transkripsiyon sonrası gen susturulması mekanizmaları tüm ökaryotlarda tespit edilmiştir şimdiye kadar okudu ve RNAi, fonksiyonel genomik çalışmalar ve diğer uygulamalar 10 için bir araç olarak hızla çeşitli organizmaların geliştirilmiştir . RNAi genetik manipülasyon için alternatif yaklaşımlar mevcut değildir ya da güvenilmez 5,11 keneler ve diğer organizmalar içinde en yaygın olarak kullanılan gen susturma tekniği haline gelmiştir. Keneler genetik karakterizasyon RNAi 12,13 son başvuru tarihine kadar sınırlı olmuştur. RNAi mevcut olduğunu kısa süre içinde, bu, karakterizasyonu ve kene patojen arayüzü tarama ve kene koruyucu antijenler 14 karakterizasyonu kene gen fonksiyonu eğitim için değerli bir araç olduğunu kanıtlamıştır. Burada, içine dsRNA beslenmemiş keneler enjeksiyon yoluyla RNAi için bir yöntem açıklanmıştır. Bu deneysel bir yaklaşım elde edilen bilgi, anlayış ve temel biyolojik sistemlerin kene istilası kontrolü ve kene kaynaklı patojenlerin 15-19 bulaşmasını önlemek için aşı geliştirme belirgin katkısı olacağı muhtemeldir .

Watch this Scientific Journal Video about RNA Interference in Ticks at JoVE.com

RNA Interference in Ticks

RNA enterferans (RNAi) dsRNA beslenmemiş keneler içine enjeksiyon için bir yöntem açıklanmıştır. RNAi genetik manipülasyon diğer yöntemlerin kullanımı sınırlı kalmıştır keneler en yaygın olarak kullanılan gen susturma tekniği.

Keneler de RNA Girişim

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Research

JoVE Journal

Immunology and Infection

18.0K Görüntüleme.  Oklahoma State University. RNA enterferans (RNAi) dsRNA beslenmemiş keneler içine enjeksiyon için bir yöntem açıklanmıştır. RNAi genetik manipülasyon diğer yöntemlerin kullanımı sınırlı kalmıştır keneler en yaygın olarak kullanılan gen susturma tekniği.

Video: RNA Interference in Ticks

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

Hücre tipi siRNA teslimat için özel anti-HIV gp120 aptamers Geliştirme

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

İmmünoloji ve Enfeksiyon

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes scapularis Keneler gelen tükürük, Tükürük Bezi ve hemolimf Koleksiyonu

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Lyme Hastalığı Araştırma İletim ve Xenodiagnosis için Hayvanlar Keneler besleme

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Fare Makrofagların yılında Doğuştan Bağışıklık Tepkisi Soruşturma RNA girişimi kullanma

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

Nucleocapsid Tavlama Dolayımlı Elektroforez (ADI) Testi HIV-1 Nükieokapsit inhibitörlerinin Hızlı tanımlanması sağlar

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

Bir uyarılabilir İfade Sistemi uygulamak Hücre içi Sinyali ile Bakteriyel Virulans Faktörler Girişim Eğitim için

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA Arıtma Hücre içindeki dan Yetiştirilen<em&gt; Listeria monocytogenes</em&gt; Makrofaj

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

Yeni Nesil Electroporasyonun tarafından İlköğretim İnsan Th17 Hücreleri Küçük RNA Transfeksiyon

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Kodlama ve RNA sınıfları kodlamayan teşhis kodu içinde domuz tam kan ifade.

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Çift iplikçikli RNA ve desen tanıma reseptörleri negatif anlamda RNA virüsü enfeksiyonu confocal görüntüleme

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...