Name: crispr/cas9 technology in restoring dystrophin expression in ipsc-derived muscle progenitors
Uploaded: 2019-09-14T12:30:00
Description: Watch this Scientific Journal Video about CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors at JoVE.com

Tang og Weintraub blev delvist støttet af NIH-AR070029, NIH-HL086555, NIH-HL134354.

Alle dyre håndterings-og operationsprocedurer blev udført ved en protokol, der er godkendt af det institutionelle udvalg for dyrepasning og-anvendelse (IACUC) i Augusta. Mus blev fodret standard kost og vand ad libitum.
1. isolering af primære mus fibroblaster fra voksne DMDMDX -mus
<ol><li>Euthanize Adult DMDMDX -mus (mand, 2 måneder gammel) ved co2 kvælning og THORACOTOMI ifølge iacuc godkendt af Medical College of Georgia, Augusta University. Skær h...

<ol>
	<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 (3), 304-313 (2012).</li><li>Batchelor, C. L., Winder, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sparks,+signals+and+shock+absorbers:+how+dystrophin+loss+causes+muscular+dystrophy.">Sparks, signals and shock absorbers: how dystrophin loss causes muscular dystrophy.</a> Trends Cell Biology. 16 (4), 198-205 (2006).</li><li>Gregorevic, 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=Systemic+delivery+of+genes+to+striated+muscles+using+adeno-associated+viral+vectors.">Systemic delivery of genes to striated muscles using adeno-associated viral vectors.</a> Nature Medicine. 10 (8), 828-834 (2004).</li><li>Bengtsson, N. E., Seto, J. T., Hall, J. K., Chamberlain, J. S., Odom, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+prospects+of+gene+therapy+clinical+trials+for+the+muscular+dystrophies.">Progress and prospects of gene therapy clinical trials for the muscular dystrophies.</a> Human Molecular Genetics. 25 (R1), R9-R17 (2016).</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 (6271), 407-411 (2016).</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 (6271), 400-403 (2016).</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 (6271), 403-407 (2016).</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=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 (6201), 1184-1188 (2014).</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> Circulation Research. 121 (8), 923-929 (2017).</li><li>Amoasii, 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=Gene+editing+restores+dystrophin+expression+in+a+canine+model+of+Duchenne+muscular+dystrophy.">Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy.</a> Science. 362 (6410), 86-91 (2018).</li><li>Eisen, 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=Electrophysiological+abnormalities+in+induced+pluripotent+stem+cell-derived+cardiomyocytes+generated+from+Duchenne+muscular+dystrophy+patients.">Electrophysiological abnormalities in induced pluripotent stem cell-derived cardiomyocytes generated from Duchenne muscular dystrophy patients.</a> Journal of Cellular and Molecular Medicine. 23 (3), 2125-2135 (2019).</li><li>Marion, R. 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=Telomeres+acquire+embryonic+stem+cell+characteristics+in+induced+pluripotent+stem+cells.">Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells.</a> Cell Stem Cell. 4 (2), 141-154 (2009).</li><li>Dumont, N. 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=Dystrophin+expression+in+muscle+stem+cells+regulates+their+polarity+and+asymmetric+division.">Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division.</a> Nature Medicine. 21 (12), 1455-1463 (2015).</li><li>Sacco, 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=Short+telomeres+and+stem+cell+exhaustion+model+Duchenne+muscular+dystrophy+in+mdx/mTR+mice.">Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice.</a> Cell. 143 (7), 1059-1071 (2010).</li><li>Su, X., 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=Purification+and+Transplantation+of+Myogenic+Progenitor+Cell+Derived+Exosomes+to+Improve+Cardiac+Function+in+Duchenne+Muscular+Dystrophic+Mice.">Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice.</a> Journal of Visualized Experiments. (146), (2019).</li><li>Su, X., 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=Exosome-Derived+Dystrophin+from+Allograft+Myogenic+Progenitors+Improves+Cardiac+Function+in+Duchenne+Muscular+Dystrophic+Mice.">Exosome-Derived Dystrophin from Allograft Myogenic Progenitors Improves Cardiac Function in Duchenne Muscular Dystrophic Mice.</a> Journal of Cardiovascular Translational Research. 11 (5), 412-419 (2018).</li><li>Ousterout, D. 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=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> Nature Communications. 6, 6244 (2015).</li><li>Barde, I., 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=Efficient+control+of+gene+expression+in+the+hematopoietic+system+using+a+single+Tet-on+inducible+lentiviral+vector.">Efficient control of gene expression in the hematopoietic system using a single Tet-on inducible lentiviral vector.</a> Molecular Therapy. 13 (2), 382-390 (2006).</li><li>Glass, K. 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Duchenne muskeldystrofi (DMD) er en destruktiv og i sidste ende dødelig arvelig sygdom karakteriseret ved en mangel på dystrofin, fører til progressiv muskelatrofi1,2. Vores resultater viser det restaurerede dystrofin-genekspression i DMDMDX IPSC-afledte myogene stamceller ved tilgangen af crispr/Cas9-medieret exon23 sletning. Denne fremgangsmåde har tre fordele.
Først genererede vi iPSCs fra DMDMDX Mouse-afl...

Duchenne muskeldystrofi (DMD) er en af de mest almindeligt muskuløse dystrophier og er karakteriseret ved fravær af dystrofin, påvirker 1 af ca 5.000 nyfødte drenge på verdensplan1. Tab af dystrofin gen funktion resulterer i strukturelle muskel defekter fører til progressive myofibers degeneration1,2. Rekombinant adeno-associeret virus (Raav)-medieret genterapi system er blevet testet for at genoprette dystrofin ekspression og forbedre muskelfunktion, såsom genudskiftning ved hjælp af mikro-dystrophiner (μ-dys). Men Raav-tilgangen kræver gentagne injektioner for at opretholde ekspression af det funktionelle protein3,4. Derfor har vi brug for en strategi, der kan give effektivt og permanent genvinde dystrofin genekspression hos patienter med DMD. DMDMDX Mouse, en musemodel til DMD, har en punkt mutation i exon 23 af dystrofin genet, der introducerer en tidlig opsigelse codon og resulterer i en ikke-funktionel trunkeret protein mangler C-Terminal dystroglycan bindende domæne. Nylige undersøgelser viste brugen af crispr/Cas9-teknologi til at genoprette dystrofin-genekspression ved nøjagtig genkorrektion eller mutant exon-sletning i små og store dyr5,6,7. Long et al.8 rapporterede metoden til korrektion af dystrofin-genmutationen i DMDMDX Mouse kimcelle ved Homology-rettet reparation (HDR) baseret crispr/Cas9 genom-redigering. El Refaey et al.9 rapporterede, at Raav effektivt kunne punktafgiftspligtige den mutante exon 23 i dystrofiske mus. I disse studier blev grnas designet i introns 20 og 23 for at forårsage dobbeltstrenget DNA-pauser, som delvist restaurerede dystrofin-ekspression efter DNA-reparation via ikke-homologe end-sammenslutning (nhej). Endnu mere spændende, amoasii et al.10 rapporterede for nylig effekten og gennemførligheden af Raav-mediated crispr gene redigering i genoprette dystrofin udtryk i canine modeller, et vigtigt skridt i fremtidig klinisk anvendelse.
DMD forårsager også stamcelle lidelser11. For muskelskader, bolig muskel stamceller genopfylde døende muskelceller efter muskel differentiering. Men, de efterfølgende cyklusser af skade og reparation føre til afkortning af telomerer i muskel stamceller12, og for tidlig udtømning af stamcelle puljer13,14. Derfor kan en kombination af autologt stamcelleterapi med genomredigering for at genoprette dystrofin ekspression være en praktisk tilgang til behandling af DMD. CRISPR/Cas9-teknologien giver mulighed for at generere autologe genetisk korrigerede inducerede pluripotente stamceller (iPSC) til funktionel muskel regenerering og forhindre yderligere komplikationer af DMD uden at forårsage immun afvisning. IPSCs har dog en risiko for tumordannelse, som kan afhjælpes ved differentieringen af iPSC til myogene stamceller.
I denne protokol beskriver vi brugen af ikke-integrerende Sendai-virus til omprogrammering af Dermal fibroblaster af DMDMDX -mus til ipscs og derefter inddrive dystrofin udtryk ved crispr/Cas9 genom sletning. Efter validering af Exon23 sletning i IPSC ved genotypebestemmelse differentierede vi Genome-korrigeret IPSC til myogenic forfædre (MPC) via myogen-induceret myogenic differentiering.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Surgical Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>31-gauge needle</td>
			<td>Various&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp Incision</td>
			<td>Various&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Scalpels</td>
			<td>Various&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Various&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast medium (for 100 mL of complete medium)</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (55 mM)</td>
			<td>Gibco</td>
			<td>21-985-023</td>
			<td>0.1 mL</td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Slution 100x</td>
			<td>CORNING</td>
			<td>MT30004CI</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium - high glucose</td>
			<td>SIGMA</td>
			<td>D6429</td>
			<td>87 mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum Characterized</td>
			<td>HyClone</td>
			<td>SH30396.03</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>L-Glutamine solution</td>
			<td>SIGMA</td>
			<td>G7513</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100x)&#160;</td>
			<td>Gibco</td>
			<td>11140076</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>TVP solution (for 500 mL of complete solution)</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Chicken Serum</td>
			<td>Gibco</td>
			<td>16110-082</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td>186 mg</td>
		</tr>
		<tr>
			<td>Phosphat-buffered saline</td>
			<td></td>
			<td></td>
			<td>to 500 mL</td>
		</tr>
		<tr>
			<td>Trypsin (2.5%)</td>
			<td>Thermo</td>
			<td>15090046</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>mES growth medium(for 500 mL of complete solution)</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (55 mM)</td>
			<td>Gibco</td>
			<td>21-985-023</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Slution 100x</td>
			<td>CORNING</td>
			<td>MT30004CI</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium - high glucose</td>
			<td>SIGMA</td>
			<td>D6429</td>
			<td>408.5 mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum Characterized</td>
			<td>HyClone</td>
			<td>SH30396.03</td>
			<td>75 mL</td>
		</tr>
		<tr>
			<td>L-Glutamine solution</td>
			<td>SIGMA</td>
			<td>G7513</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Mouse recombinant Leukemia Inhibitory Factor (LIF), 0.5 x 106 U/mL</td>
			<td>EMD Millipore Corp</td>
			<td>CS210511</td>
			<td>500 &#956;L</td>
		</tr>
		<tr>
			<td>MEK/GS3 Inhibitor Supplement</td>
			<td>EMD Millipore Corp</td>
			<td>CS210510-500UL</td>
			<td>500 &#956;L</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100x)&#160;</td>
			<td>Gibco</td>
			<td>11140076</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td colspan="4">The ES cell media should not be stored for more than 4 weeks and with inhibitors not more than 2 weeks.</td>
		</tr>
		<tr>
			<td>mES frozen medium(for 50 mL of complete solution)</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>SIGMA</td>
			<td>D2650</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium - high glucose</td>
			<td>SIGMA</td>
			<td>D6429</td>
			<td>24.9 mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum Characterized</td>
			<td>HyClone</td>
			<td>SH30396.03</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>Mouse recombinant Leukemia Inhibitory Factor (LIF), 0.5 x 106 U/mL</td>
			<td>EMD Millipore Corp</td>
			<td>CS210511</td>
			<td>50 &#956;L</td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>RRID</td>
		</tr>
		<tr>
			<td>0.05% Trypsin/0.53 mM EDTA</td>
			<td>CORNING</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Thermo scientific</td>
			<td>J19943-k2</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase solution</td>
			<td>SIGMA</td>
			<td>A6964</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>AgeI-HF</td>
			<td>NEB</td>
			<td>R3552L</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488-conjugated goat-anti-mouse antibody</td>
			<td>Invitrogen</td>
			<td>A32723</td>
			<td>AB_2633275</td>
		</tr>
		<tr>
			<td>Alexa488-conjugated goat-anti-rabbit antibody</td>
			<td>Invitrogen</td>
			<td>A32731</td>
			<td>AB_2633280</td>
		</tr>
		<tr>
			<td>Alexa555-conjugated goat-anti-rabbit antibody</td>
			<td>Invitrogen</td>
			<td>A32732</td>
			<td>AB_2633281</td>
		</tr>
		<tr>
			<td>anti-AFP</td>
			<td>Thermo scientific</td>
			<td>RB-365-A1</td>
			<td>AB_59574</td>
		</tr>
		<tr>
			<td>anti-&#945;-Smooth Muscle Actin (D4K9N) XP</td>
			<td>CST</td>
			<td>19245S</td>
			<td>AB_2734735</td>
		</tr>
		<tr>
			<td>anti-Dystrophin</td>
			<td>Thermo</td>
			<td>PA5-32388</td>
			<td>AB_2549858</td>
		</tr>
		<tr>
			<td>anti-LIN28A (D1A1A) XP&#160;&#160;&#160;</td>
			<td>CST</td>
			<td>8641S</td>
			<td>AB_10997528</td>
		</tr>
		<tr>
			<td>anti-MYH2</td>
			<td>DSHB</td>
			<td>mAb2F7</td>
			<td>AB_1157865</td>
		</tr>
		<tr>
			<td>anti-Nanog-XP</td>
			<td>CST</td>
			<td>8822S</td>
			<td>AB_11217637</td>
		</tr>
		<tr>
			<td>anti-Oct-4A (D6C8T)</td>
			<td>CST</td>
			<td>83932S</td>
			<td>AB_2721046</td>
		</tr>
		<tr>
			<td>anti-Sox2</td>
			<td>abcam</td>
			<td>ab97959</td>
			<td>AB_2341193</td>
		</tr>
		<tr>
			<td>anti-SSEA1(MC480)&#160;</td>
			<td>CST</td>
			<td>4744s</td>
			<td>AB_1264258</td>
		</tr>
		<tr>
			<td>anti-TH (H-196)</td>
			<td>SANTA CRUZ&#160;</td>
			<td>sc-14007</td>
			<td>AB_671397</td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase Live Stain (500x)</td>
			<td>Thermo</td>
			<td>A14353</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin S</td>
			<td>Sigma-Aldrich</td>
			<td>203350</td>
			<td></td>
		</tr>
		<tr>
			<td>BsmBI/Esp3I</td>
			<td>NEB</td>
			<td>R0580L/R0734L</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Millipore</td>
			<td>205805-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV&#160;</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS004189</td>
			<td></td>
		</tr>
		<tr>
			<td>Competent Cells</td>
			<td>TakaRa</td>
			<td>636763</td>
			<td></td>
		</tr>
		<tr>
			<td>CutSmart&#160;</td>
			<td>NEB</td>
			<td>B7204S</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoTune-iPS 2.0 Sendai Reprogramming Kit</td>
			<td>Thermo</td>
			<td>A16517</td>
			<td></td>
		</tr>
		<tr>
			<td>DirectPCR Lysis Reagent (cell)</td>
			<td>VIAGEN BIOTECH</td>
			<td>302-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase (1 U/mL)</td>
			<td>STEMCELL Technologies</td>
			<td>7923</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline Hydrochloride</td>
			<td>Fisher BioReagents</td>
			<td>BP26535</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI-HF</td>
			<td>NEB</td>
			<td>R3101L</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin bovine plasma</td>
			<td>SIGMA</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; 
			QIAEX II Gel Extraction Kit (500)</td>
			<td>QIAGEN</td>
			<td>20051</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin, type A</td>
			<td>SIGMA</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>HardSet Antifade Mounting Medium with DAPI</td>
			<td>Vector</td>
			<td>H-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B (50 mg/mL)</td>
			<td>Invitrogen</td>
			<td>10687010</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine HCL Injection</td>
			<td>HENRY SCHEIN ANIMAL HEALTH</td>
			<td>45822</td>
			<td></td>
		</tr>
		<tr>
			<td>KpnI-HF</td>
			<td>NEB</td>
			<td>R3142L</td>
			<td></td>
		</tr>
		<tr>
			<td>lenti-CRISPRv2-blast</td>
			<td>Addgene</td>
			<td>83480</td>
			<td></td>
		</tr>
		<tr>
			<td>lenti-Guide-Hygro-iRFP670</td>
			<td>Addgene</td>
			<td>99377</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamin 3000 Transfection Kit</td>
			<td>Invitrogen</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>LV-TRE-VP64-mouse MyoD-T2A-dsRedExpress2&#160;&#160;</td>
			<td>Addgene</td>
			<td>60625</td>
			<td></td>
		</tr>
		<tr>
			<td>LV-TRE-VP16 mouse MyoD-T2A-dsRedExpress2</td>
			<td>Addgene</td>
			<td>60626</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse on Mouse (M.O.M.) Basic Kit</td>
			<td>Vector</td>
			<td>BMK-2202</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI-HF</td>
			<td>NEB</td>
			<td>R3189L</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Media</td>
			<td>ThermoFisher</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol 4,000</td>
			<td>Alfa Aesar</td>
			<td>AAA161510B</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>SIGMA</td>
			<td>TR1003</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;BioCoat Poly-D-Lysine/Laminin Culture Slide</td>
			<td>CORNING</td>
			<td>CB354688</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerUp SYBR Green Master Mix</td>
			<td>ThermoFisher</td>
			<td>A25742</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeSTAR Max Premix</td>
			<td>TakaRa</td>
			<td>R045</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>VIAGEN BIOTECH</td>
			<td>507-PKP</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin Dihydrochloride</td>
			<td>MP Biomedicals</td>
			<td>ICN19453980</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR Lentivirus Titration Kit</td>
			<td>abm</td>
			<td>LV900</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick ligation kit</td>
			<td>NEB</td>
			<td>M2200S</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAGEN Plasmid Plus Midi Kit (100)</td>
			<td>QIAGEN</td>
			<td>12945</td>
			<td></td>
		</tr>
		<tr>
			<td>RevertAid RT Reverse Transcription Kit</td>
			<td>Thermo scientific</td>
			<td>K1691</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAzol RT</td>
			<td>Molecular Research Center, INC</td>
			<td>RN 190</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase Reaction Buffer</td>
			<td>NEB</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase</td>
			<td>NEB</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth Modified</td>
			<td>Fisher BioReagents</td>
			<td>BP9729-600</td>
			<td></td>
		</tr>
		<tr>
			<td>ViralBoost Reagent (500x)</td>
			<td>ALSTEM</td>
			<td>VB100</td>
			<td></td>
		</tr>
	</tbody>
</table>

crispr/cas9 technology in restoring dystrophin expression in ipsc-derived muscle progenitors

Duchenne muskeldystrofi (DMD) er en svær progressiv muskelsygdom forårsaget af mutationer i dystrofin genet, som i sidste ende fører til udmattelse af muskel stamceller. Grupperet jævnligt hinanden korte palindromiske gentagelser/crispr-associeret 9 (crispr/Cas9) genredigering har potentialet til at genskabe ekspression af dystrofin genet. Autologe inducerede pluripotente stamceller (iPSCs)-afledte muskelprogenitorceller (MPC) kan genopbygge stammen/stamcelle puljen, reparere skader og forhindre yderligere komplikationer i DMD uden at forårsage en immunrespons. I denne undersøgelse introducerer vi en kombination af crispr/Cas9 og ikke-integrerede IPSC-teknologier for at opnå muskel progenitorer med genvundet dystrofin-protein udtryk. Kort sagt bruger vi en ikke-integreret Sendai vektor til at etablere en iPSC-linje fra Dermal fibroblaster af DMDMDX -mus. Vi bruger derefter crispr/Cas9-sletnings strategien til at genoprette dystrofin-ekspression gennem en ikke-homologøs ende-forbindelse af det omindrammede dystrofin-gen. Efter PCR validering af exon23 udtømning i tre kolonier fra 94 plukket iPSC kolonier, vi differentiere iPSC til MPC af doxycyclin (DOX)-induceret udtryk for MyoD, en vigtig transkriptionsfaktor spiller en væsentlig rolle i reguleringen af muskel differentiering. Vores resultater viser gennemførligheden af at bruge crispr/Cas9 sletnings strategi til at genoprette dystrofin ekspression i IPSC-afledte MPC, som har betydelige muligheder for at udvikle fremtidige terapier til behandling af DMD.

Etablering af DMDMDX Skin fibroblaster afledt IPSC. Vi demonstrerede effektiviteten af at generere mus iPSCs fra DMDMDX mus afledt hud fibroblast ved hjælp af integration-fri omprogrammering vektorer. Figur 1a viste, at forekomsten af embryonale stamceller (ESC)-lignende kolonier på tre uger efter infektion. Vi evaluerer effektiviteten af iPSC induktion ved levende alkalisk fosfatase (AP) bejdsen; Figur 1b viser...

Watch this Scientific Journal Video about CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors at JoVE.com

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors

Her præsenterer vi en Cas9-baseret exon23 sletnings protokol for at genoprette dystrofin Expression i IPSC fra DMDMDX Mouse-afledte hudfibroblaster og direkte differentiere ipscs i myogenic stamceller (MPC) ved hjælp af tet-on myod aktiveringssystem.

CRISPR/Cas9-teknologi til genoprettelse af dystrophin-ekspression i iPSC-afledte muskel Progenitorer

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the ...

Duchenne er en muskelsvind forårsager for tidlig udmattelse af muskel progenitor celler. Det er en af de mest alvorlige muskelsvind. For at fremme en funktionel muskelregenerering bruger vi CRISPR/Cas9-medieret genredigering til at genoprette dystrophinudtryk i inducerede pluripotente stamceller afledte muskelkompensatorceller.CRISPR-Cas9-genredigeringen har potentiale til at genoprette dystrophin-genekspression. Autologe iPSC-afledte muskel progenitor celler kan genopbygge stamcelle pore, reparere skader, og forhindre yderligere komplikationer i DMD uden at forårsage en immunrespons. Forskellige agenter for iPS-celler i myogene progenitorceller reducerede risikoen for tumorgendannelse i iPS-celler.Flere behandlinger, der kombinerer autolog induceret Pluripotent Stem Cell-afledte muskel progenitor celler med CRISPR-Cas9-medieret genetisk korrektion er lovende Duchenne muskelsvind behandling strategier. Denne teknologi kan bruges til at behandle mange medfødte sygdomme ved genetisk redigering autologe stamceller, herunder hjerte, hjerne og blodsygdom. Vi viste demonstration af denne tilgang kan give læserne med erfaring i håndtering af celle omprogrammering og genredigering, som fortsat er en udfordring.Under et omvendt mikroskop skal du undersøge kolonierne af inficerede museembrynale stamceller. Marker kolonierne i bunden af skålen med den selvopløsende objektmarkør. Påfør smurte kloningsringe til at dække de markerede cellekolonier.Tilsæt 100 mikroliter på 0,05% Trypsin-EDTA til hver kloning ring og læg pladen i en inkubator på 37 grader Celsius i fem minutter. Derefter overføres de fordøjede celler med 100-mikroliterpipettespidser til 48-brønds kulturplader, der indeholder 250 milliliter MES vækstmedium. Inkuber cellerne i 37 Grader Celsius inkubator med 5% kuldioxid og 85% fugtighed.Når de læser 70% sammenløb, skal du vælge brønde med velvoksne celler og tilsæt 250 milliliter TVP-opløsning til at fordøje i 30 minutter. Derefter overføre cellekulturen fra hver brønd til en seks centimeter højning. Gentages anvende kloning ringe og inkubation i flere gange, indtil ensartet, dorm-formede kloner er opnået.Nu fordøje den forberedte mus iPSCs ved at tilføje en milliliter TVP løsning. Overfør cellekulturen til en 24-brøndplade belagt med fibronectin og gelatine og inkuber ved 37 grader Celsius med 5% kuldioxid. Når cellerne når op på 50 %sammenløbet, skiftes til 500 mikroliter frisk kulturmedium, der indeholder otte mikrogram pr. milliliter polybren.Derefter tilsættes 100 mikroliter af lentiviral partikelopløsning til musens iPSCs. Inkuber cellerne i tre dage ved 37 grader Celsius med 5% kuldioxid. Dernæst bestemme den mindste koncentration af Blasticidin og Hygromycin B, som er på halv dræbe sats inden for 24 timer.Tilsæt medier på 2,5 mikrogram pr. milliliter Blasticidin og 100 mikrogram pr. milliliter Hygromycin B i cellerne, vent i tre dage, og vælg de celler, der forbliver klæbende som stablingsinficerede celler. Fordøje den valgte mus iPSCs med 0,5 milliliter TVP-opløsning, hver godt i en ny 24-brønd plade, og inkubere celler i 30 minutter ved 37 grader Celsius med 5% kuldioxid. Derefter pipette iPSCs i enkelte celler og tælle cellerne med en celle optælling kammer.Vælg omkring 150 fordøjet enkelt celler og fortyndes med MES medium i 10 centimeter skål, kultur ved 37 grader Celsius med 5% kuldioxid. Efter ca. 10 dage, under et omvendt mikroskop, skal du bruge 10 mikroliter sterile pipettespidser til at plukke enkelte kolonier og overføre hver koloni til 50 mikroliter TVP-opløsning i en 96-brøndplade. Fordøje ved 37 grader Celsius i 30 minutter.Pålidelig manuel kloningsplukning er afgørende for succesen med at høste CRISPR/Cas9-redigerede inducerede pluripotente stamceller. Derefter overføre og opdele de fordøjede celler fra hver brønd i yderligere to 96-brønds kulturplader. Inkubere ved 37 grader Celsius indtil 70% sammenløb.Med cellekolonierne ved 70% sammenløb, fjerne mediet i 96-brønd plade. Der tilsættes 25 mikroliter Lysis reagens, der indeholder 1%Proteinase K-opløsning til hver brønd, og lysatet overføres til en anden 96-brønd PCR-plade. Seal PCR pladen med selvklæbende sæler og inkubere pladen i en termisk cycler ved 55 grader Celsius i 30 minutter og derefter ved 95 grader Celsius i 45 minutter til lyse celler og deaktivere Proteinase K.Derefter, i et PCR-rør, tilsæt to mikroliter af lysatet, 10 mikroliter af 2 DNAx Polymerase Premix, 7 mikrolitører af DNase-frit vand , og en mikroliter af DMD Exon23 primere.Start PCR-reaktionen i henhold til manuskriptet. Derefter indlæses den PCR-reaktion, der leveres med 2,2 mikroliter læssebuffer, på en 2% agarosegel. Kør gelen ved 100 til 150 volt i 30 minutter og undersøg gelen under UV-lys.I denne protokol blev to guide RNAs, der flankerer mutanten Exon23, designet. Efter Cas9-medieret dobbelt-strandede pauser og ikke-homologe end-sammenføjning, mutant Exon23 blev slettet, giver mulighed for afkortet, men funktionel Dystrophin produktion. For at undgå lækage af Trypsin opløsning, er vi nødt til at anvende fedt jævnt på bunden af ringene.Kombinationen af CRISPR/Cas9 genomredigering med et hæftet MyoD-aktiveringssystem giver en sikker, gennemførlig og effektiv strategi for autolog transplantation hos Patienter med muskelsvind i Duchenne med gengenforævningsceller.

Research

JoVE Journal

Developmental Biology

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Analyse af zebrafisk Larver Skeletmuskel Integritet med Evans blå farvestof

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Generering CRISPR / Cas9 Mediated monoallelisk Sletninger til Studieinformationen Enhancer funktion i mus embryonale stamceller

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Instrueret differentiering af Primitive og endelige hæmatopoietisk ophav fra humane pluripotente stamceller

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Generation af iPSC-afledte menneskelige hjerne organoider til Model Tidlig forstyrrelser i nervesystemets

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

In Vivo Imaging af transgene genekspression i Individuel Retinal progenitorer i kimære zebrafisk embryoner til Studieinformationen Cell Nonautonomous påvirkninger

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Anvendelse af RNAi og Heat-shock-induceret transskription faktor udtryk til at omprogrammere kønsceller til neuroner i C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

In Vivo Billeddannelse af muskel-sene morfogenese i Drosophila pupper

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation af stillads-fri, tre-dimensionelle Insulin udtrykker Pancreatoids fra mus i bugspytkirtlen stamfaderen In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

En in vitro 3D-model og beregnings ledning til kvantificering af det Vasculogeniske potentiale af iPSC-afledte Endotelafprogenitorer

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...