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

唐和温特劳布部分得到NIH-AR070029、NIH-HL086555、NIH-HL134354的支持。

所有动物处理和外科手术均由奥古斯塔大学机构动物护理和使用委员会（IACUC）批准的协议进行。老鼠被喂食标准饮食和水。
1. 从成年Dmdmdx小鼠中分离原虫成纤维细胞
<ol><li>根据奥古斯塔大学佐治亚医学院批准的IACUC，通过CO2窒息和胸腔切除术，将成年Dmdx小鼠（雄性，2个月大）安乐死。在层罩下无菌条件下，用无菌手术刀切割尾部。用70%乙醇冲洗尾...

<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. 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=Tissue-engineered+cartilage+with+inducible+and+tunable+immunomodulatory+properties.">Tissue-engineered cartilage with inducible and tunable immunomodulatory properties.</a> Biomaterials. 35 (22), 5921-5931 (2014).</li></ol>

杜琴肌肉萎缩症（DMD）是一种破坏性的，最终致命的遗传性疾病，其特征是缺乏营养不良症，导致渐进性肌肉萎缩1，2。我们的结果表明，通过CRISPR/Cas9介导的外显23缺失方法，Dmdmdx iPSC衍生的造血原细胞中恢复了营养不良蛋白基因表达。这种方法有三个优点。
首先，我们使用非集成RNA载体从Dmdmdx小鼠衍生的皮肤成纤...

杜氏肌营养不良症 （DMD） 是最常见的肌肉营养不良症之一，其特征是缺乏营养不良症，影响全球约 5，000 名新生儿中的 1 个 。肌营养不良基因功能的丧失导致结构肌肉缺陷，导致渐进性肌纤维退化1，2。重组腺相关病毒（rAAV）介导的基因治疗系统已经过测试，以恢复肌营养不良症的表达和改善肌肉功能，例如使用微营养不良症（μ-Dys）替换基因。然而，rAAV方法需要反复注射来维持功能蛋白3，4的表达。因此，我们需要一种策略，能够有效和永久地恢复DMD患者的营养不良症基因表达。Dmdmdx小鼠是 DMD 的小鼠模型，在肌营养不良素基因的外显子 23 中具有点突变，该基因引入过早终止通香，导致缺乏 C-端营养不良症结合域的非功能性截断蛋白。最近的研究表明，使用CRISPR/Cas9技术，通过精确的基因校正或突变外源删除在大小动物5，6，7恢复营养不良蛋白基因表达。Long等人8报告了通过同源定向修复（HDR）基于CRISPR/Cas9基因组编辑来纠正Dmdmdx小鼠生殖系中营养不良蛋白基因突变的方法。El Refaey等人9日报告说，rAAV可以有效地切除营养不良小鼠体内的突变外生23。在这些研究中，gRNA被设计在20和23的内源中，导致双链DNA断裂，通过非同源端连接（NHEJ）修复DNA后部分恢复肌营养不良症表达。更令人兴奋的是，Amoasii等人10最近报告了rAAV介导CRISPR基因编辑在恢复犬型肌营养不良蛋白表达的疗效和可行性，这是未来临床应用的重要一步。
DMD也导致干细胞紊乱11。对于肌肉损伤，住宅肌肉干细胞补充肌肉分化后死亡的肌肉细胞。然而，连续的损伤和修复周期导致肌肉干细胞12端粒缩短，干细胞池过早耗尽13，14。因此，将自体干细胞治疗与基因组编辑相结合，以恢复营养不良素表达，是治疗DMD的实用方法。CRISPR/Cas9 技术提供了产生自体基因校正诱导多能干细胞 （iPSC） 的可能性，用于功能性肌肉再生，并在不引起免疫排斥的情况下防止 DMD 的进一步并发症。然而，iPSCs有肿瘤形成的风险，可以通过iPSC分化为肌原祖细胞来缓解。
在此协议中，我们描述了使用非集成仙台病毒将Dmdx小鼠的皮肤成纤维细胞重新编程到iPSC中，然后通过CRISPR/Cas9基因组删除恢复肌营养不良素表达。通过基因分型验证iPSC中的Exon23缺失后，通过MyoD诱导的肌基分化，将基因组校正的iPSC分化为肌原原体（MPC）。

<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 肌肉营养不良症 （DMD） 是一种由肌营养不良基因突变引起的严重渐进性肌肉疾病，最终导致肌肉祖细胞衰竭。聚类定期间隔短血循环重复/CRISPR相关9（CRISPR/Cas9）基因编辑有可能恢复肌营养不良素基因的表达。自体诱导多能干细胞（iPSCs）衍生的肌肉祖细胞（MPC）可以补充干细胞/祖细胞池，修复损伤，防止DMD的进一步并发症，而不会引起免疫反应。在这项研究中，我们介绍了CRISPR/Cas9和非集成iPSC技术的组合，以获得肌祖具有恢复的营养不良蛋白表达。简单地说，我们使用非集成仙台载体从Dmdmdx小鼠的皮成纤维细胞中建立iPSC线。然后，我们使用CRISPR/Cas9删除策略，通过重新框住的营养不良基因的非同源端联接来恢复肌营养不良症表达。在PCR验证了94个精选iPSC菌落的三个菌落的外显酶消耗后，通过多氧环素（Dox）诱导的肌D表达将iPSC分化为MPC，这是一种关键转录因子，在调节肌肉分化方面起着重要作用。我们的结果表明，使用CRISPR/Cas9删除策略在iPSC衍生的MPC中恢复营养不良素表达的可行性，这为开发未来治疗DMD的疗法具有巨大潜力。

建立Dmdx皮肤成纤维细胞衍生iPSC。 我们使用无集成的重新编程载体，演示了从Dmdmdx小鼠衍生的皮肤成纤维细胞生成小鼠iPSC的效率。图1A表明，胚胎干细胞（ESC）样菌落在感染后三周出现。通过活碱性磷酸酶（AP）染色评估iPSC诱导效率;图1B显示，根据FACS分析，AP阳性细胞的百分比约为1.8%。SSEA1、Lin28、Nanog、OCT4和SOX2是?...

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

在这里，我们提出了基于Cas9的外显23删除协议，从Dmdmdx小鼠衍生的皮肤成纤维细胞恢复iPSC中的营养不良素表达，并使用Tet-on MyoD激活系统将iPSC直接分化为肌源性祖细胞（MPC）。

CRISPR/Cas9在iPSC衍生肌肉预祖中恢复肌营养不良素表达的技术

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

杜琴是一种肌肉萎缩症，导致肌肉祖细胞过早耗尽。它是最严重的肌肉萎缩症之一。为了促进功能性肌肉再生，我们使用CRISPR/Cas9培养的基因编辑来恢复诱导多能干细胞衍生肌肉祖细胞中的营养不良素表达。CRISPR-Cas9基因编辑具有恢复营养不良基因表达的潜力。自体 iPSC 衍生的肌肉祖细胞可以补充干细胞毛孔，修复损伤，并防止 DMD 的进一步并发症，而不会引起免疫反应。iPS细胞的不同代理进入肌原性原细胞，降低了肿瘤在 iPS 细胞中重新生成的风险。几种疗法结合了自体诱导多能干细胞衍生的肌肉祖细胞细胞与CRISPR-Cas9的培养基因校正是有希望的Duchenne肌肉萎缩治疗策略。这项技术可用于通过基因编辑自体干细胞（包括心脏、大脑和血液疾病）来治疗许多先天性疾病。我们展示了这种方法的演示，可以给读者提供处理细胞重新编程和基因编辑的经验，这仍然是一个挑战。在倒置显微镜下，检查受感染的小鼠胚胎干细胞的菌落。使用自墨对象标记标记菜底的菌落。涂抹润滑性克隆环以覆盖标记的细胞菌落。在每个克隆环中加入100个0.05%的 Trypsin-EDTA 微升，并将板在37摄氏度的培养箱中放置5分钟。然后，将含有100微升移液器尖端的消化细胞转移到含有250毫升 MES 生长介质的48孔培养板中。在37摄氏度的培养箱中孵育细胞，具有5%的二氧化碳和85%的湿度。当他们读取70%汇合，选择与生长良好的细胞的井，并添加250毫升的TVP溶液消化30分钟。然后，将细胞培养从每一个井转移到一个六厘米的盘子。重复应用克隆环和孵育多次，直到获得均匀的宿舍形克隆。现在，通过添加一毫升的 TVP 解决方案来消化准备好的鼠标 iPSC。将细胞培养转移到涂有纤维素和明胶的24井板中，并在37摄氏度下用5%的二氧化碳孵育。细胞达到50%汇合后，切换到500微升新鲜培养基，每毫升多纤维素含有8微克。然后，向小鼠 iPSCs 添加 100 微升的扁病毒颗粒溶液。在37摄氏度下用5%的二氧化碳孵育细胞三天。接下来，确定24小时内半杀率的布拉西丁和海格罗霉素B的最低浓度。将每毫升布拉西丁2.5微克的介质和每毫升100微克的Hygromycin B加入到细胞中，等待三天，并选择保持粘附的细胞作为稳定受感染的细胞。用0.5毫升的TVP溶液消化选定的小鼠 iPSCs，每个井都放在一个新的24井板中，并在37摄氏度下孵育细胞30分钟，二氧化碳含量为5%。然后，将 iPSC 移液到单个细胞中，然后用细胞计数室计数细胞。选择约150个消化的单细胞，用 MES 培养基稀释成 10 厘米的培养皿，培养在 37 摄氏度下，培养 5% 的二氧化碳。大约10天后，在倒置显微镜下，使用10个微升无菌移液器尖端来挑选单个菌落，并在96井板中将每个菌落转移到50微升的TVP溶液中。在37摄氏度下消化30分钟。可靠的人工克隆采摘对于成功收获CRISPR/Cas9编辑的诱导多能干细胞至关重要。然后，将每个井的消化细胞转移并分成另外两个96孔培养板。在37摄氏度下孵育，直到70%的汇合。当细胞菌落达到70%汇合时，去除96井板中的介质。将含有1%蛋白酶K溶液的25微升Lysis试剂添加到每井中，并将解酶转移到另一个96井PCR板中。用粘合密封密封 PCR 板，在 55 摄氏度的热循环器中孵育板 30 分钟，然后在 95 摄氏度下孵育 45 分钟，以熔接细胞并变性蛋白酶 K.然后，在 PCR 管中加入两个微升的脱酸酯、10 微升的 2x DNA 聚合酶预混料、7 微升无脱氧水， 和一个微升的 DMD Exon23 底板。根据手稿启动 PCR 反应。然后，将随附 2.2 微升的加载缓冲液提供的 PCR 反应加载到 2% 的 agarose 凝胶上。以 100 至 150 伏运行凝胶 30 分钟，并在紫外线下检查凝胶。在此协议中，设计了两个在突变Exon23侧侧的引导RNA。在 Cas9 调停的双链断裂和非同源最终连接被删除后，突变 Exon23 被删除，允许截断但功能性 Dystrophin 生产。为了避免 Trypsin 溶液的泄漏，我们需要将润滑脂均匀地涂抹到环的底部。CRISPR/Cas9基因组编辑与贴装的 MyoD 激活系统相结合，为具有基因恢复基质祖细胞的杜尚肌肉萎缩患者提供了安全、可行和有效的自体移植策略。

Research

JoVE Journal

Developmental Biology

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

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

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

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

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

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

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

人多能干细胞对原始和最终造血祖细胞定向分化的研究

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

源自iPSC的人脑组织体的一代新型早期神经发育障碍

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

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

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

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

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

在体内果蝇蛹肌肌腱形态的影像学研究

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

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

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

用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道

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