R.H. ABD Ulusal Sağlık hibe (R01HL116546 ve R01 AR064241) Enstitüleri tarafından desteklenir.

Bu protokol için kullanılan hayvanlar Ohio Devlet Üniversitesi laboratuvar hayvan kaynakları hayvan kullanım kılavuzlarınıza uygun olarak muhafaza. Tüm hayvan çalışmaları kurumsal hayvan bakım, kullanım ve Ohio State Üniversitesi İnceleme Komitesi tarafından yetkili.
1. tasarım ve gRNAs CRISPR vektör içine klonlama
<ol><li>2 gRNAs dystrophin intron 20 ve intron 23 (i20 ve i23) SaCas9 için hedefleme seçin: i20: 5'-GGGCGTTGAAATTCTCATTAC CAGAGT ve i23: (PAM sıra altı çizili ve italik) 5'-CACCGATGAGAGGGAAAGGTC CTGAAT . Not: Hedef için i20 ve i23 hakkında 23 kilo baz çifti (kbp) uzak sitelerdir. Kodlama bir gen bozmaya, SaCas9 için iki gRNAs NNGRRT PAM ile ortak ekzonlar hedefleme sıra (tercihen NNGAAT, NNGGGT ve NNGAGT) ve 20 kbp mesafesinde önerilir.</li>
	<li>Arabellek tavlama x 1 gRNA oligos (100 µmol/L) tavlamak için aşağıdaki PCR parametrelerini kullanın (0,5 mol/L K-asetat, 150 mmol/L HEPES tampon stok tavlama x 5 içerir-KOH, 10 mmol/L Mg-asetat, pH7.4): 95 ° C 10 dk 59 95 ° C 0.4 ° C ile 90 döngüleri azaltma başına döngüsü için 20 s, 59-32 ° C 20 devir başına 0.3 ° C azalma ile 90 döngüleri s, 32-26 ° C 20 devir başına 0.3 ° C azalma ile 20 döngüleri s.</li>
	<li>Tavlanmış gRNA oligos pX601-CMV-SaCas9-mPA-U6-gRNA ligate BsaI ve T4 DNA ligaz bir tepki olarak kullanarak (1 µL 50 ng/µL pX601-CMV-SaCas9-mPA-U6-gRNA, 1 µL komplementer oligos, 1,5 µL 10 x T4 DNA ligaz tampon, 0.5 µL T4 DNA ligaz, 1 µL BsaI, 10 µL GKD2O ), PCR cycler aşağıdaki reaksiyon koşulu: [37 ° C 5 dk ve 16 ° C 10 dk]; 5 döngüleri 37 ° C 20 dk; 80 ° C 5 dk.</li>
	<li>15 µL ligasyonu ürün 30 µL yetkili hücrelere dönüşümü, agar plaka ile 100 µg/mL ampisilin ve kültür için 24 h 37 ° C'de hücrelerde plaka.</li>
	<li>5-10 kolonileri ve Kültür 100 µg/mL ampisilin 37 ° c, 3-4 h için bir shaker kuluçka 250 d/d içeren LB ortamda almak.</li>
	<li>Koloniler tarafından PCR ekran: 10 µL PCR ana mix, 8 µL GKD2O, 1 µL E. Coli kültür, 1 µL 10 µmol/L U6 ileri astar, 1 µL i20 veya i23 gRNA ters astar x 2. PCR Bisiklete binme parametreleri: 95 ° C 5 dk, 95 ° C 30 s, 61 ° C 30 s, 72 ° C 30 s 35 döngüleri.</li>
	<li>5 mL kültür pozitif klonların ekleyerek 4 mL taze LB orta containing100 µg/mL ampisilin ve kültür gecede 37 ° C'de, 250 d/d hazırlayın.</li>
	<li>Plazmid DNA uygun plazmid ekstraksiyon kiti kullanarak arındırmak ve Sanger tarafından plazmid doğrulayın U6 ileri astar ile sıralama.</li>
	<li>%10 FBS ve % 1 ile DMEM hücrelerde C2C12 kültür kalem-Strep plazmid etkinliğinin düzenleme gen testleri için ~ %70 confluency ulaşan kadar.</li>
	<li>Electroporate 5 µg SaCas9/gRNA plazmid karışımı 1:1 molar oranı 1 x 106 C2C12 hücrelere üreticinin protokolüne göre toplam.</li>
	<li>48 saat sonrası transfection sonra C2C12 hücre hasat ve genomik DNA ayıklayın.</li>
	<li>Fare dystrophin odağı için PCR reaksiyonu gerçekleştirmek için kullanım 100 ng genomik DNA şablon olarak: ileri astar 5'-GGCCAAAGCAAACTCTGGTA (1 µL 10 µmol/L), ters astar 5'-TTTAATCCCACGTCATGCAA (1 µL 10 µmol/L), 10 µL 2 x yeşil PCR master mix, 7 µL ddH2O, 1 µL genomik DNA () 100 ng). Kullanmak PCR Bisiklete binme koşulları 95 ° C 5 dk 35 döngüsü [95 ° C 30 s, 61 ° C 30 s, 72 ° C 30 s], 72 ° C 2 dk.</li>
</ol>2. rAAV üretim
<ol><li>Hücre kültür için AD293 hücreleri 20-40 cm 15 cm yemekleri üzerine tohum ve %10 FBS ve % 1 ile DMEM korumak kalem-Strep ~ %90 confluency transfection için ulaşan kadar.</li>
	<li>Seyreltik 3 Plasmid'ler ile 200 µL serum Orta (çanak) başına azaltılmış: 1) 10 µg pHelper; 2) 5 µg pXR (rh.74 veya diğer serotipleri) ve 3) 5 µg karışımı pX601 i20 ve pX601-i23 (1:1 oran) toplam. Polyethylenimine (PEI) transfection 80 µL PEI ile karıştırılarak gerçekleştirmek ve 10 dk. Ekle AD293 hücre kültürleri üzerine karışımı oda sıcaklığında dropwise bir şekilde kuluçkaya.</li>
	<li>72 h sonrası transfection sonra AAV virüs üretim için medya ve AD293 hücre topakları Santrifüjü 1100 x g 10 dakika için de tarafından toplamak için bir hücre kazıyıcı kullanın.</li>
	<li>Kuluçka sıvı azot ve 42 ° C su banyosu içinde alternatif ile 3 donma-çözülme çevrimleri ve ardından hücre topakları 15 mL rAAV lizis arabelleği (50 mmol/L Tris Bankası, 150 mmol/L NaCl, pH 8,5) resuspend. Hücre lysates onlar hemen işlenmeyen Eğer gelecekte işlenmesi için-80 ° C'de depolayın.</li>
	<li>Kültür medya 0.45 µm ile filtre ve %40 PEG8000% 8 son bir konsantrasyon için 2.5 mol/L NaCl ekleyin. 1s için 4 ° C'de karışımı ve 1100 x g, santrifüj 15dk için kuluçkaya.</li>
	<li>Granül rAAV lizis arabellekte resuspend ve benzonase (50 U/mL) tedavisi için 1 h 37 ° C'de ve ardından adım 2.3, hücre lysates ile birleştirir.</li>
	<li>Karışım 1100 x g 4 ° C'de 15 dakika içeren benzonase tedavi rAAV santrifüj kapasitesi ve süpernatant (ham rAAV hazırlık) kaydedin.</li>
</ol>3. rAAV arıtma ve toplama
<ol><li>Şu sırada bir ultracentrifuge tüp iodixanol degradede üzerine Ham rAAV hazırlık en baştan altına yük: ham AAV lysate (~ 15 mL), 8 mL % 15 Iodixanol, 6 mL % 25 Iodixanol, 5 mL % 40 Iodixanol, 5 mL % 58'i Iodixanol. 1 x DPBS kullanarak üst tüp boş kalan hacmi doldurun.</li>
	<li>230,000 x g ultracentrifuge Open End 120 dk 10 ° c de örnekler santrifüj kapasitesi ve 3-4 mL 18 G iğne ile rAAV parçacıkları içeren % 40 degrade fraksiyonunun toplamak.</li>
	<li>1 x DPBS ve santrifüj filtre ünitesi (MWCO 100 kDa) kullanarak santrifüj ile rAAV parçacıklar sulandırmak. İçin bu adımı yineleyin 3 - 5 kez ve nihayet rAAV hazırlık için 300-500 µL konsantre.</li>
</ol>4. rAAV Titering
<ol><li>2 konsantre µL rAAV, etanol içinde 3 M NaOAc ve resuspend 20 µL GKD2O. ile acele genomik DNA ayıklamak</li>
	<li>RAAV Nefelometri için standart eğri pX601 plazmid seri seyreltme tarafından elde edilir: 1 x 109, 1 x 108,1 x 107, 1 x 106, 1 x 105 kopya her 10 µL GKD2O. Dilute DNAase tedavi rAAV örnek olarak 1:10, 1:50, 1: 100 ve 1: 1000.</li>
	<li>QPCR seti üreticisinin yönerge göre bir gerçek zamanlı PCR sistemi kullanarak rAAV titresi nicel gerçek zamanlı PCR (qPCR) tarafından belirlemek: 1 µL 10 µmol/L her astar (5'-GGATTTCCAAGTCTCCACCC ve 5'-TCCCACCGT ACACGCCTAC), 5 µL PCR ana karışımı, 1 µL seyreltilmiş x 2 rAAV örnekleri ve 2 µL GKD2O. çalıştırmak reaksiyon aşağıdaki Bisiklete binme parametrelerle: 95 ° C 3 dk, [95 ° C 5 sn ve 61 ° C 20 sn] 45 döngüleri.</li>
</ol>5. Intravenous ve rAAVrh.74-CRISPR Neonatal mdx/utrophin+ /- fareler içine mayi enjeksiyon
<ol><li>1 dk. için buz yerleştirerek mdx/utrophin+ /- fare pups (3 gün) hareketsiz ve 1 x 1012 viral parçacıkların bir retro-orbital yaklaşım veya bir mayi enjeksiyon yoluyla sistemik fare başına 50 µL ile yönetmek.</li>
	<li>Fareyi kurtarmak ve geri ev onların kafes yerleştirmek izin. On hafta sonra rAAV yönetim, fareler CO2 maruz doku koleksiyonu için ötenazi.</li>
	<li>Ek-dokular için genomik DNA ve RNA ayıklama ve kullanma isopentane en uygun kesme sıcaklık kurcalayarak cryosectioning için bileşik dondurmak için sıvı azot soğutma donma.</li>
</ol>6. sonuçlar düzenleme hedef gen analizi
<ol><li>
Genomik DNA PCR Analizi
	<ol>
<li>Mdx/utrophin+ /- fareler kalp dokuları genomik DNA ile veya olmadan rAAV tedavi toplam yalıtmak.</li>
		<li>Aynı iletişim kuralını adım 1.12 PCR çözümleme için kullanın.</li>
	</ol>
</li>
	<li>
RT-PCR analiz dystrophin ifade
	<ol>
<li>Toplam RNA üreticisinin takip RNA ekstraksiyon kiti ile kalp dokulardan ayıklayın.</li>
		<li>Ters transkripsiyon gerçekleştirmek için önceden bir RNase free DNaz ile toplam RNA tedavi ve tedavi RNA'ın 5 µg Ters transkripsiyon kiti ile ilk iplikçikli cDNA sentezi için şablon olarak kullanmak.</li>
		<li>RT-PCR dystrophin ifade dystrophin cDNA astar ile çözümlenmesi için Ters transkripsiyon ürün kullanın: 5'-GGCTAGAGTATCAAACCAACATCAT ve 5'-TGGAGGCTTACGGTTTTATCC.</li>
	</ol>
</li>
	<li>
QPCR analizi ile etkinlik düzenleme gene tahmin
	<ol>
<li>Tarafından kantitatif RT-PCR ileri astar (exon 21) kullanarak exon 22 içeren transkript azalma tespit etmek için etkinlik düzenleme gene tahmin: 5'-TTGTCAGCTCTTCAGCCTCAAA ve ters astar (exon 23): 5'-AAACTCTCCTTCACAGTGTCACT. Gapdh bir başvuru gen ileri astar ile kullanın: 5'-AGGTTGTCTCCTGCGACTTC ve ters astar: 5'-GGTGGTCCAGGGTTTCTTACT.</li>
		<li>Nicel gerçek zamanlı PCR (qPCR) qPCR seti üreticisinin yönerge göre 2-adım reaksiyon koşulları ve gerçek zamanlı PCR sistemi kullanarak çalıştırın: 95 ° C 3 dk, 95 ° C 5 sn ve 61 ° C 30 sn 45 döngüleri.</li>
	</ol>
</li>
	<li>
Ayirt dystrophin ifade analiz etmek için boyama
	<ol>
<li>Bir cryostat bir kalınlık-in 10 µm. düzeltme kullanarak donmuş doku dondurulmuş bölümleri ile 15 dakika oda sıcaklığında %4 paraformaldehyde kesti.</li>
		<li>2 x PBS ile yıkama ve 1 h için çözüm (% 10 at serum) engelleme ile kuluçkaya.</li>
		<li>Gecede 4 ° C'de anti-dystrophin birincil antikor kuluçkaya.</li>
		<li>PBS ile slaytlar yıkama dakika 3 x 5 ve 1 h için oda sıcaklığında floresan ikincil antikorlar (1:500) ile kuluçkaya.</li>
		<li>Montaj orta DAPI ile kullanarak ve bir ters confocal mikroskopla Imaging slaytlar bağlayın.</li>
	</ol>
</li>
</ol>7. kapalı-hedef analiz T7E1 tahlil tarafından
<ol><li>Online CRISPR kullanarak potansiyel hedef kapalı siteleri tasarım araçları gibi tahmin < http://crispr.mit.edu>.</li>
	<li>5-10 potansiyel hedef kapalı siteleri PCR tarafından üst kapsayan genomik bölgeleri yükseltmek: genomik DNA 200 ng, 1 µL yüksek-sadakat DNA polimeraz, 5 µL 10 x PCR arabellek karıştırmak, 1,5 µL 10 µmol/L, siteye özgü ileriye ve geriye doğru astar ile 50 µL için toplam ses düzeyini ayarlama GKD2O.</li>
	<li>PCR ürünleri Elektroforez tarafından çalıştırmak, hulâsa ve koşmak bir jel ekstraksiyon kiti kullanılarak DNA arındırmak. Spektrofotometreler kullanarak toplama ölçmek.</li>
	<li>Denaturing ve yavaş yavaş bir PCR cycler kullanarak arabellek 2.1 içinde saf amplicons yeniden tavlama tarafından heteroduplex oluşumu teşvik. 1.2. adımda açıklandığı gibi aynı Bisiklet koşullarını kullanın.</li>
	<li>Yeniden tavlanmış ürünler için 0.5 µL T7E1 enzim ile 37 ° C'de 30 dk sindirmek ve reaksiyon kullanarak % 1-2 özel jel elektroforez tarafından analiz. Not: Amplicons da hedeflenen derin sıralama tarafından analiz edilebilir.</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eteplirsen.">Eteplirsen.</a> Hosp Pharm. 52, 302-305 (2017).</li><li>Sicinski, P., 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=The+molecular+basis+of+muscular+dystrophy+in+the+mdx+mouse:+a+point+mutation.">The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.</a> Science. 244, 1578-1580 (1989).</li><li>Xu, L., 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=CRISPR-mediated+Genome+Editing+Restores+Dystrophin+Expression+and+Function+in+mdx+Mice.">CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice.</a> Molecular therapy : the journal of the American Society of Gene Therapy. 24, 564-569 (2016).</li><li>El Refaey, 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=In+Vivo+Genome+Editing+Restores+Dystrophin+Expression+and+Cardiac+Function+in+Dystrophic+Mice.">In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice.</a> Circ Res. 121, 923-929 (2017).</li><li>Tabebordbar, 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=In+vivo+gene+editing+in+dystrophic+mouse+muscle+and+muscle+stem+cells.">In vivo gene editing in dystrophic mouse muscle and muscle stem cells.</a> Science. 351, 407-411 (2016).</li><li>Pozsgai, E. R., Griffin, D. A., Heller, K. N., Mendell, J. R., Rodino-Klapac, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Sarcoglycan+gene+transfer+decreases+fibrosis+and+restores+force+in+LGMD2E+mice.">beta-Sarcoglycan gene transfer decreases fibrosis and restores force in LGMD2E mice.</a> Gene therapy. 23, 57-66 (2016).</li><li>Chicoine, L. G., 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=Plasmapheresis+eliminates+the+negative+impact+of+AAV+antibodies+on+microdystrophin+gene+expression+following+vascular+delivery.">Plasmapheresis eliminates the negative impact of AAV antibodies on microdystrophin gene expression following vascular delivery.</a> Mol Ther. 22, 338-347 (2014).</li><li>Chicoine, L. G., 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=Vascular+delivery+of+rAAVrh74.MCK.GALGT2+to+the+gastrocnemius+muscle+of+the+rhesus+macaque+stimulates+the+expression+of+dystrophin+and+laminin+alpha2+surrogates.">Vascular delivery of rAAVrh74.MCK.GALGT2 to the gastrocnemius muscle of the rhesus macaque stimulates the expression of dystrophin and laminin alpha2 surrogates.</a> Mol Ther. 22, 713-724 (2014).</li><li>Vouillot, L., Thelie, A., Pollet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+T7E1+and+surveyor+mismatch+cleavage+assays+to+detect+mutations+triggered+by+engineered+nucleases.">Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases.</a> G3. 5, 407-415 (2015).</li><li>Tsai, S. Q., 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=GUIDE-seq+enables+genome-wide+profiling+of+off-target+cleavage+by+CRISPR-Cas+nucleases.">GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.</a> Nat Biotechnol. 33, 187-197 (2015).</li></ol>

Hiçbir çıkar çatışması bulunmaktadır.

Bu protokol için biz vivo içinde kalp gen rAAVrh.74-aracılı teslim SaCas9 ve iki gRNAs kullanma Doğum sonrası farelerde düzenleme elde etmek için gerekli tüm adımları ayrıntılı olarak. Yukarıda belirtildiği, bu yaklaşım bizim iş27' açıklandığı gibi dystrophin ifade distrofik farelerde geri yüklemek için kullanılabilir, ancak Ayrıca hızlı ters genetik çalışmalar uzun süreci olmadan farelerde kalp ile ilgili genlerin sağlayabilirsiniz geleneksel nakavt yak...

Kümelenmiş, düzenli olarak interspaced, kısa palindromik tekrar (CRISPR) teknolojisi en son ilerleme genom Mühendisliği temsil eder ve mevcut uygulama hücrelerinde ve organizmaların genetik devrim yarattı. Genom CRISPR tabanlı düzenleme kullanır CRISPR ilişkili bir endonükleaz CRISPR ilişkili protein 9 (Cas9) gibi yönlendirmek için tek bir rehber RNA (gRNA) ve Cpf1 protospacer için sırayla tamamlayıcı bir genomik DNA hedef için kodlanmış ile gRNA tarafından bir belirli protospacer bitişik motifi (PAM)1,2. PAM Cas9 veya Cpf1 geni bakteriyel türler bağlı olarak değişir. Streptococcus pyogenes (SpCas9) elde edilen en sık kullanılan Cas9 nükleaz sigara hedef iplikçikteki genomik DNA doğrudan hedef dizisi aşağı akım bulunur NGG PAM dizisi tanır. Hedef sitede, sonra homolog olmayan sonu (NHEJ) katılma ve Homoloji yönetmen onarım (HDR) yolları3de dahil olmak üzere iç hücresel DNA tamir mekanizmaları tamir iki iplikçikli sonu (DSB), Cas9 ve Cpf1 proteinleri oluşturmak, 4. Homolog rekombinasyon DNA şablon yokluğunda, DSB öncelikle eklemeleri veya silmeleri (indels) ile sonuçlanabilir hataya NHEJ yolu tarafından DSB kodlama oluşturulmuşsa frameshift mutasyonlar neden nükleotit, onarılana bölge bir gen5,6. Böylece, CRISPR sistem yaygın olarak işlev kaybı mutasyonlar çeşitli hücresel modelleri ve tüm organizmalar, mühendis bir gen işlevi araştırmak amacıyla kullanılmaya başlanmıştır. Ayrıca, catalytically etkin olmayan Cas9 ve Cpf1 için transcriptionally geliştirilmiştir ve epigenetically hedef genlerin7,8ifade modüle.
Daha yakın zamanlarda, SpCas9 ve SaCas9 ( Staphylococcus aureustüretilmiş) Doğum sonrası gen düzeltme, insan hastalıkları, hayvan modeli için rekombinant adeno ilişkili virüs (rAAV) vektörel çizimler içine paketlenmiş özellikle, Duchenne kas distrofisi (DMD)9,10,11,12,13,14,15. DMD dystrophin protein16,17-kodlar, DMD gen mutasyonlar neden bir ölümcül X'e bağlı resesif yenidoğan tarama18 göre yaklaşık bir 3500 erkek doğumlular etkileyen bir hastalıktır , 19. progresif kas güçsüzlüğü ve israf ile karakterizedir. DMD hastaları genellikle 10 ile 12 yaş ve solunum ve/veya kalp yetmezliği ölmektedir arasında 20-30 yıl20yaşına yürüyecek yeteneğini kaybetmezsin. En belirgin olarak kardiyomiyopati gelişen > %90 DMD hastaları ve DMD hastaları21,22ölüm önde gelen neden temsil eder. Her ne kadar Deflazacort (anti-inflamasyon ilaç) ve Eteplirsen (bir exon ilacı exon 51 atlama atlama) son zamanlarda DMD için gıda ve İlaç Dairesi (FDA)23,24tarafından bu tedavilerin hiçbiri onaylanmıştır genomik DNA düzeyinde genetik kusur düzeltmek. Bir nokta mutasyon exon 23 dystrophin gen taşır, mdx fare DMD modeli25yaygın olarak kullanmıştır. Ayrıca, biz daha önce fonksiyonel dystrophin ifade çerçeve silme ekzonlar 21, 22 ve 23 Kas içi reklam-SpCas9/gRNA teslim9 kullanarak mdx farelerin iskelet kasları'kapsayan genomik DNA'ın tarafından restore edilerek gösterdi ve intravenöz ve mayi rAAVrh.74-SaCas9/gRNA teslim26kullanarak mdx/utrophin+ /- fareler Kalp kaslarının.
Bu protokol için dystrophin rAAVrh.74-SaCas9/gRNA vektörler, kalp kas bölümlerde dystrophin analiz gRNA tasarım kullanarak mdx/utrophin+ /- farelerde geri yüklemek için Doğum sonrası kardiyak gen düzenleme her adımı biz ayrıntılı olarak tarif.

<table>
	<tbody>
		<tr>
			<td>Alexa Fluor 555 goat anti-rabbit IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21428</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio Safety Cabinets</td>
			<td>Thermo Fisher Scientific</td>
			<td>1347</td>
			<td>1300 Series A2 Class II, Type A2</td>
		</tr>
		<tr>
			<td>BsaI</td>
			<td>New England BioLabs Inc</td>
			<td>RO535S</td>
			<td></td>
		</tr>
		<tr>
			<td>Competent cells</td>
			<td>Agilent Technologies</td>
			<td>200314</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dishes, 150mm</td>
			<td>Nest Scientific USA</td>
			<td>715001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>Leica CM3050S</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>MT10017CV</td>
			<td>Corning cellgro</td>
		</tr>
		<tr>
			<td>Dystrophin antibody</td>
			<td>Spring Bioscience</td>
			<td>E2660</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast-Ion Midi Plasmdi Kits</td>
			<td>IBI Scientific</td>
			<td>IB47111</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>26-140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Unit, 0.45&#956;m</td>
			<td>Thermo Fisher Scientific</td>
			<td>166-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>GoTaq Master Mixes</td>
			<td>Promega</td>
			<td>M7122</td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed plasmid mini kit</td>
			<td>IBI Scientific</td>
			<td>IB47102</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Abcam</td>
			<td>ab139501</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp 2239 Water Bath</td>
			<td>Thermo Fisher Scientific</td>
			<td>15-460-11Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp Heat Block</td>
			<td>Thermo Fisher Scientific</td>
			<td>11-718</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>Carl Zeiss Microscopy, LLC</td>
			<td></td>
			<td>LSM 780</td>
		</tr>
		<tr>
			<td>LightCycler 480 instrument</td>
			<td>Roche</td>
			<td>5015243001</td>
			<td>LightCycler 480 Instrument II</td>
		</tr>
		<tr>
			<td>Large Capacity Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>46-910</td>
			<td>Thermo Scientific Sorvall RC 6 Plus</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002445</td>
			<td>Sorvall Legend Micro 21R</td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometers</td>
			<td>Thermo Scientific</td>
			<td>ND2000CLAPTOP</td>
			<td>NanoDrop&#8482; 2000/2000c</td>
		</tr>
		<tr>
			<td>Oligos for i20-F</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>CACCgGGCGTTGAAATTCTCATTAC</td>
		</tr>
		<tr>
			<td>Oligos for i20-R</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>AAAC GTAATGAGAATTTCAACGCCc;</td>
		</tr>
		<tr>
			<td>Oligos for i23-F</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>CACCgCACCGATGAGAGGGAAAGGTC</td>
		</tr>
		<tr>
			<td>Oligos for i23-R</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>GACCTTTCCCTCTCATCGGTGc</td>
		</tr>
		<tr>
			<td>Oligos</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep Density Gradient Medium</td>
			<td>Sigma-Aldrich</td>
			<td>D1556-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG8000</td>
			<td>Sigma-Aldrich</td>
			<td>89510-1kg-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Polysciences</td>
			<td>23966</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>P2308</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick-Seal centrifuge tube</td>
			<td>Beckman Coulter Inc</td>
			<td>342414</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Radiant SYBR Green Hi-ROX qPCR Kits</td>
			<td>Alkali Scientific</td>
			<td>QS2005</td>
			<td></td>
		</tr>
		<tr>
			<td>RevertAid RT Reverse Transcription Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K1691</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotor</td>
			<td>Beckman Coulter Inc</td>
			<td>337922</td>
			<td>Type 70Ti</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England BioLabs Inc</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Bio-rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter Inc</td>
			<td>8043-30-1192</td>
			<td>Optima le-80k</td>
		</tr>
		<tr>
			<td>Ultra centrifugal filter unit</td>
			<td>MilliporeSigma</td>
			<td>UFC510096</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Mounting Medium with DAPI</td>
			<td>Vector Laboratories, Inc</td>
			<td>H-1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

adeno-associated virus-mediated delivery of crispr for cardiac gene editing in mice

Kümelenmiş, düzenli olarak interspaced, kısa, palindromik tekrar (CRISPR) sistemi büyük ölçüde kültürlü hücrelerinde ve canlıların çeşitli türlerin genom mühendislik kolaylaştırdı. CRISPR teknoloji aynı zamanda insan hastalıkların bir dizi roman therapeutics olarak keşfedilmeyi. Kanıtı-of-concept veri tarafından fizibilite ve gen düzenleme tabanlı tedavi yaklaşımı Duchenne kas distrofisi (DMD) bir fare modeli kullanarak için etkinliği gösteren çalışmalar olarak son derece cesaret verici. Özellikle, rekombinant adeno ilişkili virüs (rAAV) serotip rh.74 (rAAVrh.74) intravenöz ve mayi enjeksiyon Staphylococcus aureus CRISPR ilişkili protein 9 verimli bir şekilde kalp teslim (SaCas9) ve iki sağladı exon 23 fare Dmd gen mutasyona uğramış bir kodon ile genomik bir bölgeyi silmek için (gRNA) RNA'ların yol. Bu aynı yaklaşım gen-in-faiz vurmak ve ne zaman gRNA gen Kodlayıcı bölge hedeflemek için tasarlanmıştır kendi kardiyak fonksiyon postnatal farelerde çalışma için kullanılabilir. Bu protokol için rAAVrh.74-CRISPR vektör mühendis ve yüksek verimli kardiyak teslim yenidoğan farelerde elde ayrıntılı olarak gösterir.

Hedef genomik DNA bölgenin silinmesi ikna etmek için gRNAs etkinliği hücre kültürlerinde ambalaj içinde vivo çalışmalar için rAAV içine önce değerlendirilmelidir. Bu amaçla, gRNAs ve SaCas9 ifade yapıları electroporated C2C12 hücre içine ve genomik DNA PCR tarafından hedef siteleri (Şekil 1) kanat astar ile analiz edildi. ~ 500 kan basıncı kontrol hücreleri genomik DNA'ın büyük boyutları nedeniyle bir grup verir iken gen düzenleme...

Watch this Scientific Journal Video about Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice at JoVE.com

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

Burada vivo içinde kalp gen rekombinant Adeno-Associated virüs (rAAV) kullanarak farelerde düzenleme taşımak için detaylı bir protokol sağlamak-CRISPR teslimini aracılı. Bu iletişim kuralı distrofik kardiyomiyopati Duchenne kas distrofisi tedavisinde umut verici bir tedavi stratejisi teklifler ve kardiyak özgü nakavt postnatal farelerde oluşturmak için kullanılabilir.

Virüs-aracılı teslim CRISPR kardiyak Gene farelerde düzenleme için Adeno ilişkili

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

adeno-associated-virus-mediated-delivery-crispr-for-cardiac-gene

Research

JoVE Journal

Medicine

8.1K Görüntüleme.  Ohio State University Wexner Medical Center. Burada vivo içinde kalp gen rekombinant Adeno-Associated virüs (rAAV) kullanarak farelerde düzenleme taşımak için detaylı bir protokol sağlamak-CRISPR teslimini aracılı. Bu iletişim kuralı distrofik kardiyomiyopati Duchenne kas distrofisi tedavisinde umut verici bir tedavi stratejisi teklifler ve kardiyak özgü nakavt postnatal farelerde oluşturmak için kullanılabilir.

Video: Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

Rekombinant Adeno Associated Virus serotip 3 Vektörler Karaciğer Kanseri Hücreleri Yüksek Verimlilik İletimi

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Tıp

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Bağışıklık Eksik Fare Kuruluş ve İnsan Retinoblastom Tümörler yayılımı

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Kardiyak Stres Testi Dobutamin İndüklemiş ve Farelerde Kalp Kateterizasyonu tarafından İzlenen

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

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 ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

Farelerde Safra Kanalı ligasyonu: Obstrüktif Kolestaz tarafından İnflamatuar Karaciğer Hasarı ve Fibrozis indüksiyonu

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Aracı Olarak Adeno-ilişkili virüs kullanarak Hastalığında Retina Engelleri Eğitim için

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

Bir rekombinant adeno-ilişkili viral vektörün stereotaktik enjeksiyon yoluyla Parkinson Hastalığı için bir alfa-sinüklein Bazlı Fare Modeli Geliştirme

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Yenidoğan Farelerde Miyokard Enfarktüsü, Kalp Rejenerasyon Bir Model

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss. In this study, we describe the posterior semicircular canal approach as a viable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear. The easy anatomic identification of the posterior semicircular canal, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery.

In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear.

İç kulak gen teslim yenidoğan Mouse için posterior semisirküler kanal yaklaşım

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.