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. er støttet af U.S. National Institutes sundhed tilskud (R01HL116546 og R01 AR064241).

De dyr, der anvendes i denne protokol blev opretholdt ved The Ohio State University laboratorium animalsk ressourcer i overensstemmelse med retningslinjer for brug af dyr. Alle dyreforsøg blev godkendt af institutionelle Animal Care, brug og bedømmelsesudvalg i Ohio State University.
1. design og kloning af gRNAs i CRISPR vektor
<ol><li>Vælg 2 gRNAs målretning dystrofin intron 20 og intron 23 (i20 og i23) for SaCas9: i20: 5'-GGGCGTTGAAATTCTCATTAC CAGAGT og i23: 5'-CACCGATGAGAGGG...

<ol>
	<li>Makarova, K. S., Koonin, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annotation+and+Classification+of+CRISPR-Cas+Systems.">Annotation and Classification of CRISPR-Cas Systems.</a> Methods Mol Biol. 1311, 47-75 (2015).</li><li>Zetsche, B., 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=Cpf1+is+a+single+RNA-guided+endonuclease+of+a+class+2+CRISPR-Cas+system.">Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.</a> Cell. 163, 759-771 (2015).</li><li>Cong, 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=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339, 819-823 (2013).</li><li>Mali, 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=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339, 823-826 (2013).</li><li>Lieber, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+double-strand+DNA+break+repair+by+the+nonhomologous+DNA+end-joining+pathway.">The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway.</a> Annual review of biochemistry. 79, 181-211 (2010).</li><li>Ran, F. 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=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature protocols. 8, 2281-2308 (2013).</li><li>Zhang, X., Wang, J., Cheng, Q., Zheng, X., Zhao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+gene+regulation+by+CRISPR-ddCpf1.">Multiplex gene regulation by CRISPR-ddCpf1.</a> Cell discovery. 3, 17018 (2017).</li><li>Zhang, X. H., Tee, L. Y., Wang, X. G., Huang, Q. S., Yang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Off-target+Effects+in+CRISPR/Cas9-mediated+Genome+Engineering.">Off-target Effects in CRISPR/Cas9-mediated Genome Engineering.</a> Molecular therapy. Nucleic acids. 4, e264 (2015).</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> Mol Ther. 24, 564-569 (2015).</li><li>Long, C., 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=Postnatal+genome+editing+partially+restores+dystrophin+expression+in+a+mouse+model+of+muscular+dystrophy.">Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy.</a> Science. 351, 400-403 (2016).</li><li>Long, C. 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. R., Lloyd-Puryear, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+of+MDA+muscle+disease+symposium+on+newborn+screening+for+Duchenne+muscular+dystrophy.">Report of MDA muscle disease symposium on newborn screening for Duchenne muscular dystrophy.</a> Muscle Nerve. 48, 21-26 (2013).</li><li>Spurney, C. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+in+the+therapy+of+cardiomyopathy+in+muscular+dystrophy.">New approaches in the therapy of cardiomyopathy in muscular dystrophy.</a> Annual review of medicine. 58, 75-88 (2007).</li><li>Baker, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approvals,+Submission,+and+Important+Labeling+Changes+for+US+Marketed+Pharmaceuticals.">Approvals, Submission, and Important Labeling Changes for US Marketed Pharmaceuticals.</a> Hosp Pharm. 52, 306-307 (2013).</li><li>Baker, D. 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>

I denne protokol detaljer vi alle de skridt, der er nødvendige for at nå i vivo hjerte gen redigering i postnatal mus ved hjælp af rAAVrh.74-medieret levering af SaCas9 og to gRNAs. Som tidligere nævnt, denne fremgangsmåde kan bruges til at gendanne dystrofin udtryk i dystrofe mus som beskrevet i vores arbejde27, men det kunne også aktiverer hurtig omvendt genetik undersøgelser af hjerte-relaterede gener i mus uden den langvarige proces med den traditionelle knockout tilgang til at...

De klynger, regelmæssigt interspaced, korte palindromiske Gentag (CRISPR) teknologi repræsenterer den seneste avancement i genom engineering og har revolutioneret den nuværende praksis med genetik i celler og organismer. CRISPR-baseret genom-redigering anvender en enkelt guide RNA (gRNA) for at lede en CRISPR-associerede liv1975 såsom CRISPR-forbundet protein 9 (Cas9) og Cpf1 til genomisk DNA mål, der supplerer i sekvens til protospacer kodet af gRNA med en specifikke protospacer tilstødende motiv (PAM)1,2. PAM varierer afhængigt af den bakterielle arter af Cas9 eller Cpf1-genet. De mest almindeligt anvendte Cas9 nukleasen afledt af Streptococcus pyogenes (SpCas9) genkender en PAM sekvens af NGG, der findes direkte neden for target sekvens i genomisk DNA, på ikke-målarter strand. Mål-webstedet genererer Cas9 og Cpf1 proteiner en dobbelt-strenget pause (DSB), som derefter repareret via iboende cellulære DNA reparation mekanismer, herunder ikke-homologe ende sammenføjning (NHEJ) og homologi-instrueret reparation (HDR) veje3, 4. I mangel af en DNA-skabelon til homolog rekombination, er DSB primært repareret af den fejlbehæftede NHEJ vej, hvilket kan resultere i indrykninger eller sletninger (indels) af nukleotider forårsager frameshift mutationer, hvis DSB blev oprettet i kodning region af en gen5,6. Således, CRISPR systemet har været meget anvendt til ingeniør tab af funktion mutationer i forskellige cellulære modeller og hele organismer, for at undersøge funktionen af et gen. Derudover katalytisk inaktive Cas9 og Cpf1 er blevet udviklet til transcriptionally og modulere epigenetically udtryk for target gener7,8.
Mere nylig, både SpCas9 og SaCas9 (afledt af Staphylococcus aureus) har været pakket ind i rekombinant adeno-associeret virus (rAAV) vektorer for postnatal gen korrektion i dyremodel af sygdomme hos mennesker, især, Duchenne muskeldystrofi (DMD)9,10,11,12,13,14,15. DMD er en fatal X-linked recessive sygdomme skyldes mutationer i DMD -genet, som koder dystrofin protein16,17, der påvirker omkring én i 3500 mandlige fødsler ifølge nyfødte screening18 , 19. det er karakteriseret ved fremadskridende muskelsvækkelse og spilde. DMD patienter miste normalt evnen til at gå mellem 10 og 12 år og dø af luftvejssygdomme og/eller hjerte svigt i en alder af 20-30 år20. Navnlig, kardiomyopati udvikler i > 90% af DMD patienter og repræsenterer førende forårsage død i DMD patienter21,22. Selv om Deflazacort (en anti-inflammation medicin) og Eteplirsen (en exon overspringelse medicin for overspringning exon 51) har for nylig blevet godkendt til DMD af Food and Drug Administration (FDA)23,24, ingen af disse behandlinger rette den genetiske defekt på genomisk DNA-niveau. MDX-mus, som bærer et punkt mutation på exon 23 i dystrophin genet, har været meget anvendt som en DMD model25. Desuden demonstreret vi tidligere, at det funktionelle dystrofin udtryk blev restaureret af-frame sletning af genomisk DNA dækker exons 21, 22 og 23 i skeletmuskulatur af mdx mus ved hjælp af intramuskulær Ad-SpCas9/gRNA levering9 , og i hjertet muskler af mdx/utrophin+ /- mus ved hjælp af intravenøs og intraperitoneal rAAVrh.74-SaCas9/gRNA levering26.
I denne protokol beskriver vi i detaljer hvert trin i postnatal hjerte gen redigering til at gendanne dystrofin i mdx/utrophin+ /- mus ved hjælp af rAAVrh.74-SaCas9/gRNA vektorer, fra gRNA design dystrofin analyse i afsnittene hjerte muskel.

<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

Grupperet, regelmæssigt interspaced, kort, palindromiske Gentag (CRISPR) systemet har høj grad lettet genom engineering i både dyrkede celler og levende organismer fra en lang række arter. CRISPR-teknologi er også blevet undersøgt som roman therapeutics for en række af sygdomme hos mennesker. Proof-of-concept data er yderst opmuntrende, som eksemplificeret af nylige undersøgelser, der viser gennemførligheden og effektiviteten af genet redigering-baserede terapeutisk tilgang til Duchenne muskeldystrofi (DMD) ved hjælp af en murine model. Især har intravenøs og intraperitoneal injektion af rekombinant adeno-associeret virus (rAAV) serotype rh.74 (rAAVrh.74) aktiveret effektiv hjerte levering af Staphylococcus aureus CRISPR-forbundet protein 9 (SaCas9) og to guide RNA'er (gRNA) for at slette en genomisk region med en mutant codon i exon 23 af musen Dmd -genet. Denne samme fremgangsmåde kan også bruges til at udskære gen af interesse og studere deres hjertefunktion i postnatal mus når gRNA er designet til at målrette den kodende region af genet. I denne protokol viser vi i detaljer hvordan du ingeniør rAAVrh.74-CRISPR vektor og hvordan man kan opnå meget effektiv hjerte levering i neonatal mus.

Effekten af gRNAs til at fremkalde sletning af målområde for genomisk DNA bør evalueres i cellekulturer før emballage i rAAV for in vivo-undersøgelser. Med henblik herpå, gRNAs og SaCas9-udtrykker konstruktioner var electroporated i C2C12 celler og genomisk DNA blev analyseret af PCR med primere flankerende målwebsteder (figur 1). En PCR produkt på ~ 500 bp angiver vellykkede sletning af den indskyde genomisk DNA fra gen redigering mens kontrol celler...

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

Her giver vi en detaljeret protokol til at udføre i vivo hjerte gen redigering i mus ved hjælp af rekombinante Adeno-Associated Virus (rAAV)-medieret levering af CRISPR. Denne protokol tilbyder en lovende terapeutisk strategi for at behandle dystrofe kardiomyopati i Duchenne muskeldystrofi og kan bruges til at generere hjerte-specifikke knockout i postnatal mus.

Adeno-associeret Virus-medieret levering af CRISPR for hjerte gen redigering i mus

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

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

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

Produktion og Påvisning af Reactive Oxygen Species (ROS) i kræft

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

Cardiac Stress Test induceret af Dobutamin og overvåges af hjertekateterisation i mus

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 ...

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

Bile Duct Ligering i mus: Induktion af inflammatorisk leverskade, og fibrose ved Obstruktiv Kolestase

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

Brug Adenoassocieret virus som værktøj til at studere Retinale Barrierer i 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 ...

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

Udvikling af en Alpha-synuclein Based rottemodel for Parkinsons sygdom via Stereotaktisk injektion af et Rekombinant adenoassocieret viral vektor

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

Myokardieinfarkt i neonatale mus, en model af 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 ...

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

Bageste halvrunde kanalen tilgang til indre øre gen levering i Neonatal mus

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 ...