Bu çalışma Hedef odaklı Ar-Ge, Japonya Bilim ve Teknoloji Ajansı, Japonya Özel Okullar Tanıtım ve Yardımlaşma Corporation Bilim Araştırma Teşvik Fonu aracılığıyla Uyarlanabilir ve Dikişsiz Teknoloji Transferi Programı tarafından desteklenen ve JSPS KAKENHI Hibe Numaraları 24300342 ve 15H04313 oldu .

Bir heptamer tipi 1. İnşaat sgRNA Kütüphanesi <ol><li> Tam ölçekli bir kütüphane inşa edilecek sürece 16.384 (4 7) 7-nt dizilerinin bir alt kümesini seçin. Not: dizileri rasgele ve / veya belirli bir hücre RNA&#39;lar hedeflemek için tasarlanmış olabilir. İkincisi, saç tokası yapıları 4,10 hemen alt baş 7-nt dizilere tamamlayıcı uygun bir bilgisayar programı ve / veya görsel olarak basıp dizilerin yardımı ile, bir hedef RNA tRNA T kolunu benzer saç tokası yapıları bulmak 17,18. </li><li> Seçilen heptamerlerin her sentezleme tam 2&#39;- O metillenmiş, 5&#39;- ve bir DNA / RNA sentezleyici ile 3&#39;-fosforile edilmiş RNA. Kontrollü gözenekli cam (CPG) Zemin üzerinde fosforamidit yöntemini kullanarak, ve 5&#39;-terminal 4,4&#39;-dimetoksitritil (DMT) sentezin son döngüde (5 &#39;fosfat ilave) koruma grubu terk 23. NOT: Özel synthetic RNA da oligonükleotid tedarikçilerden elde edilebilir. </li><li> Son sentez döngüsünün tamamlanmasından sonra, 8 saat boyunca 55 ° C &#39;de, 1 ml 29%&#39; si amonyum hidroksit ekleyin Teflon astar vidalı kapaklı bir cam şişeye sütun sentezlenen oligonükleotid ile CPG transferi ve inkübe CPG, oligonükleotidin ayrılması ve bazlar ve fosfatlar koruma gruplarını kaldırmak. </li><li> 40 ° C &#39;de santrifüj lü bir evaporatörde amonyum hidroksit buharlaştınn ve 0.2 ml 0.1 M n-hexylammonium asetat içinde oligonükleotid yeniden çözülür. </li><li> % 10-50 asetonitril / 0.1 M n-hexylammonium asetat ihtiva eden bir tampon maddesi ile bir polistiren-divinilbenzen kolonu kullanılarak ters-faz yüksek performans sıvı kromatografi yoluyla sentetik oligonükleotit saflaştınlır. 80 ° C de 25 dakika süreyle / 2.0 ml dk sütun çalıştırın. </li><li> % 20 asetik asit solüsyonu yapmak için kısımlara ayrılmış örnek (1-2 mi) konsantre asetik asit 0.25-0.5 ml ilave edilir, ve doğmuş iyileşmesinite oda sıcaklığında 1 saat boyunca bu çözelti, DMT grubu çıkarmak için. </li><li> 40 ° C &#39;de santrifüj lü bir evaporatörde örnek kurutun, 1 mi 29% amonyak içinde yeniden çözülür ve 5&#39; fosfat yan zincirini çıkarmak için oda sıcaklığında 15 dakika boyunca çözeltiden inkübe edin. </li><li> 40 ° C &#39;de santrifüj lü bir evaporatörde 0.3 ml numune hacmi (1 mi) azaltır. </li><li> kesme 1000, 4 ° C&#39;de bir gece boyunca moleküler ağırlığa sahip bir diyaliz tüpü kullanılarak damıtılmış su karşı tüm konsantre edildi örneği (500 mi) diyaliz. </li><li> Daha sonra, bir pipet ile 1.5 ml tüp diyaliz tüpünden diyaliz örnek aktarın 40 ° C &#39;de santrifüj lü bir evaporatörde kurutun ve hacmi arasında olmalıdır, su için enjeksiyon dereceli su içinde yeniden çözülür daha büyük bir 100 uM çözelti yapmak için yeteri kadar küçük olabilir. </li><li> bir spektrofotometre ile 260 nm&#39;de RNA numunesinin optik yoğunluğunun ölçülmesi ve 100 olan konsantrasyonunu ayarlamakSu için enjeksiyon dereceli su ilave edilerek uM. -20 ° C Kullanmadan önce aşağıda RNA çözümü saklayın. </li></ol> Hücre Canlılığına için sgRNA Kütüphanesi 2. Eleme <ol><li> Hazırlama 10 3 hücre / 99 ul / göz (ör HL60 ve RPMI-8226),% 10 fetal sığır serumu ve% 1 penisilin-streptomisin çözeltisi ile takviye edilmiş RPMI-1640 ortamı içinde bir 96-yuvalı tabak üzerinde. Sadece arka plan çıkarma için ortam içeren kuyu hazırlayın. numune kuyuları çevreleyen kuyu boş steril su ekleyin, kenar etkilerini ortadan kaldırmak için. </li><li> her bir suda için enjeksiyon dereceli su içinde çözülmüş heptamer tipi sgRNA (100 uM) 1 ul ekle. üç kez, her sgRNA deneyi. 3 gün boyunca% 5 CO2 nemlendirilmiş inkübatör içinde 37 ° C&#39;de inkübe plakası. </li><li> Her bir oyuğa, bir ticari olarak temin edilebilir WST-8 çözeltisinin uygun bir hacmi ekleyin. 1 ila 4 saat boyunca, aynı inkübatörde 37 ° C&#39;de inkübe plakası. </li><li> absorbans a ölçünBir mikro levha okuyucusu ile 450 nm t. NOT: Bir çok durumda, emme ve canlı hücre sayısı arasındaki doğrusal absorbans değeri 1.0 aşmadıkça muhafaza gibi görünmektedir. </li></ol> Akış Sitometrisi ile Etkili sgRNAlardır 3. Değerlendirilmesi <ol><li> Hazırlama 10 3 hücre /% 10 fetal bovin serumu ve% 1 penisilin-streptomisin çözeltisi ile takviye edilmiş RPMI-1640 ortamı içinde bir 96-yuvalı tabak üzerinde 99 ul / göz (ör HL60). bir sgRNA test edilecek en az 10 kuyu hazırlayın. numune kuyuları çevreleyen kuyu boş steril su ekleyin, kenar etkilerini ortadan kaldırmak için. </li><li> her bir suda için enjeksiyon dereceli su içinde çözülmüş heptamer tipi sgRNA (100 uM) 1 ul ekle. 3 gün boyunca% 5 CO2 nemlendirilmiş inkübatör içinde 37 ° C&#39;de inkübe plakası. </li><li> bir pipet, bir 5-ml&#39;lik bir tüp içine en az 10 kuyu aynı sgRNA ile muamele hücreleri toplamak. 30 ° C&#39;de bir santrifüj içinde 5 dakika boyunca tüp spin0 xg ve medya atın. </li><li> tüpe 1X fosfat tamponlu salin (PBS) 2 ml ilave edilir ve hücreler yeniden askıya. 300 x g&#39;de santrifüj içinde 5 dakika boyunca tüp spin ve PBS atın. tüp 1x anneksin V bağlayıcı tampon 150 ul ekleyin ve hücrelerin yeniden askıya. </li><li> , Fikoeritrin 2 ul (PE) ile konjüge edilmiş anneksin V çözeltisi ve tüp 7-aminoactinomycin D (7AAD) çözeltisi 1 ul ekle iyice karıştırın ve karanlıkta, oda sıcaklığında 15 dakika boyunca tüp inkübe edin. 24 debi sitometresinde örnek analiz edin. </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cytosolic+tRNase+ZL+can+downregulate+gene+expression+through+miRNA.">Human cytosolic tRNase ZL can downregulate gene expression through miRNA.</a> FEBS Lett. 583, 3241-3246 (2009).</li><li>Ninomiya, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+small+guide+RNAs+for+tRNase+ZL+from+human+plasma,+peripheral+blood+mononuclear+cells,+and+cultured+cell+lines.">Potential small guide RNAs for tRNase ZL from human plasma, peripheral blood mononuclear cells, and cultured cell lines.</a> PLoS One. 10, 0118631 (2015).</li><li>Takahashi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+a+heptamer-type+sgRNA+library+for+potential+therapeutic+agents+against+hematological+malignancies.">Screening of a heptamer-type sgRNA library for potential therapeutic agents against hematological malignancies.</a> Leukemia Res. 38, 808-815 (2014).</li><li>Shivarov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRUE+gene+silencing+for+hematologic+malignancies.">TRUE gene silencing for hematologic malignancies.</a> Leukemia Res. 38, 729 (2014).</li></ol>

Niigata University of Pharmacy and Applied Life Sciences has a pending patent application on a subset of the heptamer-type sgRNAs.

In most cases, fully 2&#8242;-O-methylated, 5&#8242;- and 3&#8242;-phosphorylated heptamer-type sgRNAs dissolved in water-for-injection-grade water (in the concentration of 100 &#181;M) appear to be stable at below -20 &#176;C for at least one year, judging from their activity to induce apoptosis in cancer cells. However, their stability would change depending on their sequence, quality, and purity. In some cases, 5&#8242;- or 3&#8242;-phosphate of a part of sgRNA molecules appears to be removed spontaneously during storage, judging from mass spectrometry analysis, although this may not affect their activity seriously.
When a hit sgRNA is obtained by the library screening, its cellular target RNAs can be found by transcriptome analysis through a DNA microarray and/or next-generation sequencing. The data obtained so far, however, suggest that downregulation levels of target RNAs via heptamer-type sgRNAs are usually moderate regardless of the presence or absence of transfection reagents10,12,14,17,18. 
The advantage of heptamer-type sgRNAs targeting BCL2 and WT1 mRNAs is that they can efficiently induce apoptosis in human leukemia cells in spite of weak downregulation of the target mRNAs17,18, suggesting the presence of additional RNA targets and/or additional mechanisms other than TRUE gene silencing. 
Since even the screening of the library containing only 156 heptamer-type sgRNAs can make 20 hits, around 2,000 hits would be expected to be obtained by screening the full-scale library composed of 16,384 heptamers22,25. 
For future applications, sgRNAs other than the heptamer type can be more suitable for TRUE gene silencing11-13,15,16, although heptamer-type sgRNAs are easier, more accurate and cheaper to synthesize than longer sgRNAs. Once the therapeutic sgRNA candidate is obtained, it is easy to proceed to animal model experiments.

(TR Nase Z L sonra adlandırılan - u E fficacious gen susmasının tilizing) DOĞRU gen susturma RNA yönelik gen susturma teknolojileri 1 biridir. Bu teknoloji geliştirdi tRNase Z L (ya da 3 &#39;tRNase), tRNA 3&#39; işleme endoribonuclease özelliklerine dayanarak olmuştur: Bir tanıyarak yapay küçük bir rehber RNA (sgRNA) yönetimindeki herhangi beklenen yerinde herhangi bir hedef RNA yarabilir 9 - hedef RNA ve sgRNA 2 arasında oluşturulmuş önceden tRNA benzeri ya da mikro-ön-tRNA-benzeri bir yapı. uzunluğunda nt genellikle 7-31 olan sgRNA, dört gruba, 5&#39;-yarım-tRNA, heptamer RNA, 14-nt lineer RNA ve kanca RNA kategorize edilir. 15 - Biz hücreler 10 canlı içine çeşitli yapay olarak tasarlanmış sgRNAlardır sunarak DOĞRU gen susturma etkinliğini göstermiştir. Biz de sgRNAlardır hücrelerin wi tarafından alınabilir göstermiştirherhangi bir transfeksiyon reaktifleri thout ve hedef RNA seviyelerini bozulduğunu ve / veya insan kanser hücrelerinin 16 apoptosis doğurabileceği - 18. 21 - DOĞRU gen susturma tRNase Z L ve doğal sgRNAlardır 19 üzerinden hücre içi ve hücreler arası gen düzenleyici ağ sistemi üzerinde çalışması için görünür. Kan kanserleri 22 potansiyel terapötik heptamer tipi sgRNAlardır aramak için 156 heptamer tipi sgRNAlardır içeren bir sgRNA kütüphane inşa ettik. İnsan miyeloma ve lösemi hücrelerinin ömrü üzerindeki etkisi için filtrelenmiş ve 20 tanesi etkin kanser hücre çizgilerinin en az biri apoptosis bulmuşlardır. Ve bunların 4 fare ksenogreft deneylerinde tümör büyüme oranlarını azaltmak için gösterilmiştir. Burada kan kanserlerine karşı potansiyel terapötik ilaçların bir heptamer tipi sgRNA kitaplığının taranması için bir protokol mevcut. protokol nasıl dahilhücre canlılığı ve nasıl daha fazla flow sitometri ile etkili sgRNAlardır değerlendirmek için her sgRNA etkisini değerlendirmek için nasıl sgRNA kütüphanesini oluşturmak için. Bunlar, bu protokolde yer almaz, ancak fare ksenogreft modellerinde sgRNA hücresel hedef RNAlar için analiz ve etkili sgRNAlardır değerlendirilmesi, geleneksel yöntemlerle 17,22 göre gerçekleştirilebilir.

<table border="0" cellpadding="0" cellspacing="0" width="948">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Custom heptamer RNA</td>
			<td style="width:167px;">Nippon Bioservice</td>
			<td style="width:144px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OligoSep Prep HC Cartridge</td>
			<td style="width:167px;">Transgenomic</td>
			<td style="width:144px;">99-3860</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Water-for-injection-grade Water</td>
			<td style="width:167px;">Otsuka Pharmaceutical&#160;</td>
			<td style="width:144px;">7131400A2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI-1640 medium</td>
			<td style="width:167px;">Wako</td>
			<td style="width:144px;">189-02025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">172012-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Penicillin-Streptomycin Mixed Solution</td>
			<td style="width:167px;">Nacalai Tesque</td>
			<td style="width:144px;">26253-84</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 Well Plate</td>
			<td style="width:167px;">Greiner Bio-one</td>
			<td style="width:144px;">655 180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Counting Kit-8</td>
			<td style="width:167px;">DOJINDO</td>
			<td style="width:144px;">343-07623</td>
			<td>WST-8 solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate Reader, Sunrise Thermo RC-R</td>
			<td style="width:167px;">TEKAN</td>
			<td style="width:144px;">510-82851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS Round-Bottom Tube</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:144px;">60819-820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">P7059-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Annexin V Binding Buffer</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">51-66121E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Annexin V</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">51-65875X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7AAD</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">A9400-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Cytometer, FACSCalibur</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">342973</td>
			<td></td>
		</tr>
	</tbody>
</table>

true gene silencing: screening of a heptamer-type small guide rna library for potential cancer therapeutic agents

DOĞRU gen susturma (tR Nase Z L sonra adlandırılan - u, e fficacious gen susturulması tilizing) tRNA 3 &#39;işleme endoribonuclease, tRNase Z L rehberlik edecek bir yapay küçük bir rehber RNA (sgRNA) kullanır teknolojileri susturma RNA yönettiği genin, biri bir hedef RNA tanımak için. sgRNAlardır bir transfeksiyon reaktifleri olmayan hücreler tarafından alınabilir ve hedef RNA seviyelerini bozulduğunu ve / veya insan kanser hücrelerinde apoptosisi teşvik edebilir. İnsan miyeloma ve lösemi hücrelerinin canlılığı üzerindeki etki için 156 heptamer tipi sgRNAlardır ihtiva eden bir sgRNA kitaplığı tarandı ve bunların 20 verimli, kanser hücre çizgilerinin en az biri apoptosis bulmuşlardır. Burada kan kanserlerine karşı potansiyel terapötik ilaçların bir heptamer tipi sgRNA kitaplığının taranması için bir protokol mevcut. protokol hücre canlılığı ve her sgRNA etkisini değerlendirmek nasıl fu için nasıl, sgRNA kütüphane oluşturmak için nasıl dahilrther flow sitometri ile etkili sgRNAlardır değerlendirir. Yaklaşık 2000 hit 16.384 heptamerlerin oluşan tam ölçekli sgRNA kütüphanenin taranması ile elde edilebilir olması beklenmektedir.

Altı heptamer tipi sgRNAlardır 5&#39;-AUCUUCA-3 &#39;(H1885), 5&#39;-ACACACA-3&#39; (H3277), 5&#39;-GGGGGCG-3 &#39;(H10927), 5&#39;-GGGGCCC-3&#39; (H10944), 5&#39;-GCCCCCG-3 &#39;(H12287), ve 5&#39;-CACCAGC-3&#39; (H13260) kimyasal 5&#39;- ve 3&#39;-fosfat ihtiva eden 2&#39;-O-metil RNA sentez edildi. Bu sgRNAlardır insan lösemi hücre hattı, HL60, ve bir insan miyeloma hücre hattı RPMI-8226 canlılığı üzerindeki etkileri incelendi. Hücre canlılığı deneylerinin Örnek neden olur. Şekil 1 &#39;de dört sgRNAlardır H1885, H3277, H10927 gösterilir ve H13260 çok etkili ve sırası ile&gt;% 80 ve&gt;% 70 ile HL60 ve RPMI-8226 hücre canlılığı azaltılmış. heptamerlerin H3277 ve H10927 de kanserli olmayan HEK293 hücre canlılığı üzerindeki etkileri bakımından test edildi, ve neredeyse Affe gösterilmiştirct hücre 22 canlılığı. Annexin V ve 7AAD çift boyama ile akış sitometrisi heptamer H1885, H3277, H10927 veya H13260 ile muamele HL60 hücreleri için gerçekleştirilmiştir. Her sgRNA, erken ve geç apoptotik aşamalarında toplam hücre sayısı (56-95%) sahte (Şekil 2) bu (% 4-6) daha yüksek oldu. Bu gözlemler, HL60 hücre canlılığının azaltılması apoptoza bağlı olduğuna işaret etmektedir. <img alt="Şekil 1" src="/files/ftp_upload/53879/53879fig1.jpg" /> Şekil 1. Hücre canlılığı ölçülür. Hücre canlılığı, HL60 hücrelerinde (A) veya RPMI-8226 hücreleri (B) yokluğunda ya da çıplak heptamer tipi sgRNA H1885 1 uM, H3277, H10927 mevcudiyetinde kültürlenmiştir 72 saat sonra ölçüldü H10944, H12287 veya H13260. sgRNAlardır yokluğunda göreceli canlı hücre sayıları 100 Hata ayarlanırçubuklar SD gösterir (n = 3). * P &lt;0.002. Lösemi Araştırma, Vol yayımlanmaktadır. 38. Takahashi, M. ve diğ., Hematolojik maligniteler karşı potansiyel tedavi aktif maddeleri, s 808-815, Şek bir heptamer tipi sgRNA kütüphanesinin taranması. 1, Telif Hakkı (2014), Elsevier izniyle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53879/53879fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="şekil 2" src="/files/ftp_upload/53879/53879fig2.jpg" /> Şekil 2. Akış sitometrisi. HL60 hücreleri ile ilgili olarak, akış sitometrisi Annexin V ve 7AAD çift boyama yapıldı. Hücreler yokluğunda ya da çıplak heptamer tipi sgRNA H1885 (A), H3277 (A) H13260 (A) ya da H10927 (B) 1 uM varlığında kültürlenen 72 saat sonra analiz edilmiştir. Lösemi Araştırma, Vol yayımlanmaktadır. 38, Takahashi, M. ve diğ., Hematolojik maligniteler karşı potansiyel tedavi aktif maddeleri, s 808-815, Şek bir heptamer tipi sgRNA kütüphanesinin taranması. 2, Telif Hakkı (2014), Elsevier izniyle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53879/53879fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents at JoVE.com

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

Burada kan kanserlerine karşı potansiyel terapötik ilaçların bir heptamer tipi sgRNA kitaplığının taranması için bir protokol mevcut.

DOĞRU gen susturma: Potansiyel Kanser Tedavi Ajanlar için heptamer tipi Küçük Rehberi RNA Kütüphanesi Taranması

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

true-gene-silencing-screening-heptamer-type-small-guide-rna-library

Nanyang Technological University

Research

JoVE Journal

Medicine

5.6K Görüntüleme.  Niigata University of Pharmacy and Applied Life Sciences.  Burada kan kanserlerine karşı potansiyel terapötik ilaçların bir heptamer tipi sgRNA kitaplığının taranması için bir protokol mevcut. 

Video: TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

Bronş Thermoplasty: Şiddetli Astım Yeni Bir Tedavi Yaklaşımı

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

Tıp

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

İnsan Takip edilen-Lusiferaz Malign Periferik Sinir Kılıf Tümör Hücreleri Ortotopik Xenografting In vivo</emAday Terapötik Ajanlar> Test

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

Fare intraserebroventriküler sayesinde Terapötik Ajanlar teslimi (ICV) ve intravenöz (IV) Enjeksiyon

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

Basit Fare İntrakranial Xenograftlarında Çalışmaları Kılavuzu Vida Yöntemi

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

Kanser gen Fonksiyon Eğitim için DNA vektör tabanlı RNA Girişim

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Bir Fetal Fare Modelinde Biyoaktif Ajan intratrakeal Yönetimi İçin Yeni Bir Cerrahi Yaklaşım

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Bir Zebra balığı Otokton Tümör Modeli kullanılarak Melanom Değiştiricileri için Tarama

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Gene Teslim Çalışmaları Ortotopik Mesane Kanseri Modeli

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Kültür Hücreler Kanser ile ilişkili Sinyal İletimi sorgulanması için bir Multiplexed Lusiferaz tabanlı Tarama Platformu

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Formation Odak: bir hücreye dayalı tahlil, bir genin onkojenik potansiyeli belirlemek için

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...