Name: adeno-associated virus-mediated delivery of crispr for cardiac gene editing in mice
Uploaded: 2018-08-02T13:00:00
Description: Watch this Scientific Journal Video about Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice at JoVE.com

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...

<ol>
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Z., 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=Prevention+of+muscular+dystrophy+in+mice+by+CRISPR/Cas9-mediated+editing+of+germline+DNA.">Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA.</a> Science. 345, 1184-1188 (2014).</li><li>Nelson, C. E., 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+improves+muscle+function+in+a+mouse+model+of+Duchenne+muscular+dystrophy.">In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.</a> Science. 351, 403-407 (2016).</li><li>Ousterout, D. G. K., Thakore, A. M., Majoros, P. I., Reddy, T. E. W. H., Gersbach, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+CRISPR/Cas9-based+genome+editing+for+correction+of+dystrophin+mutations+that+cause+Duchenne+muscular+dystrophy.">Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy.</a> Nat Commun. 6, 6244 (2015).</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>Bengtsson, N. E., 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=Muscle-specific+CRISPR/Cas9+dystrophin+gene+editing+ameliorates+pathophysiology+in+a+mouse+model+for+Duchenne+muscular+dystrophy.">Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.</a> Nat Commun. 8, 14454 (2017).</li><li>Hoffman, E. P., Brown, R. H., Kunkel, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin:+the+protein+product+of+the+Duchenne+muscular+dystrophy+locus.">Dystrophin: the protein product of the Duchenne muscular dystrophy locus.</a> Cell. 51, 919-928 (1987).</li><li>Nowak, K. J., Davies, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duchenne+muscular+dystrophy+and+dystrophin:+pathogenesis+and+opportunities+for+treatment.">Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment.</a> EMBO reports. 5, 872-876 (2004).</li><li>Mendell, J. R., 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=Evidence-based+path+to+newborn+screening+for+Duchenne+muscular+dystrophy.">Evidence-based path to newborn screening for Duchenne muscular dystrophy.</a> Annals of neurology. 71, 304-313 (2012).</li><li>Mendell, J. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyopathy+of+Duchenne+muscular+dystrophy:+current+understanding+and+future+directions.">Cardiomyopathy of Duchenne muscular dystrophy: current understanding and future directions.</a> Muscle Nerve. 44, 8-19 (2011).</li><li>Moriuchi, T., Kagawa, N., Mukoyama, M., Hizawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autopsy+analyses+of+the+muscular+dystrophies.">Autopsy analyses of the muscular dystrophies.</a> Tokushima J Exp Med. 40, 83-93 (1993).</li><li>McNally, E. 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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. 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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 ...

Research

JoVE Journal

Medicine

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

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 ...

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

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 ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Production and Detection of Reactive Oxygen Species ROS in Cancers

Kanserler Üretim ve reaktif oksijen türlerinin (ROS Algılama)

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

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

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

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

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

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

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

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 ...