利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞
Summary
本协议详细描述了在无饲养条件下从人类胰腺细胞中生成无足迹诱导多能干细胞 (干细胞), 其次是使用 CRISPR/Cas9 均一进行编辑和修饰单细胞克隆。
Abstract
胚胎和诱导多能干细胞可以自我和分化成多个细胞类型的身体。因此, 多能细胞在再生医学研究中备受青睐, 目前正处于眼部疾病、糖尿病、心脏病和其他疾病的临床试验中。分化成专门的细胞类型的潜能, 加上基因组编辑技术的最新进展, 包括 CRISPR/Cas 系统, 为不同应用的 iPSC 基因组的剪裁提供了更多的机会包括疾病建模, 基因治疗, 和偏微分的途径, 命名为少数。在可用的编辑技术中, 来自链球菌化脓的 CRISPR/Cas9 已成为 site-specific 编辑真核基因组的首选工具。CRISPRs 是易于访问, 低廉, 高效的工程目标编辑。该系统需要一个 Cas9 核酸和一个引导序列 (20) 特定的基因组目标邻接一个3核苷 “NGG” protospacer 相邻主题 (PAM) 的目标 Cas9 到期望的基因组所在地, 旁边的通用 Cas9 结合示踪 RNA (一起被称为单一指南 RNA 或 sgRNA)。在这里, 我们提出了一个 step-by-步骤协议, 有效地生成馈线无关和无足迹 iPSC 和描述方法的基因组编辑 iPSC 使用 Cas9 核糖 (RNP) 配合物。基因组编辑协议是有效的, 可以很容易地复用 pre-complexing sgRNAs 为多个目标与 Cas9 蛋白, 同时传递到细胞。最后, 我们描述了一个简化的方法, 以识别和表征干细胞与所需的编辑。两者结合在一起, 预计将简化 iPSC 的生成和编辑, 以用于多种应用。
Introduction
通过重编程因子的过度表达, 将人体细胞重新编程为多能状态, 使干细胞研究在疾病建模、再生医学和药物开发等领域有了革命性的改变。一些病毒重新编程的方法可以提供重新编程的因素和生成干细胞, 但该过程是劳动密集型, 而不是非常有效的1。病毒方法虽然很有效, 但却与集成和瘤2、34的问题有关。在这篇手稿中, 我们报告了使用胞质仙台病毒提供重新编程的因素和建立无足迹 iPSC 线, 没有任何病毒载体序列集成到他们的基因组5。仙台病毒是一种 RNA 病毒, 被稀释出来的细胞细胞质〜10通道感染后, 产生了丰富的重编程因素, 导致快速和有效的重新编程6,7。建立的干细胞可以很容易地转换为无饲养介质, 以避免使用小鼠胚胎成纤维细胞 (MEFs) 作为饲养细胞8。
在本刊物中, 除了概述仙台病毒介导的重新编程, 我们还描述了一个改进的编辑干细胞的协议, 它有可能提供无限的人类细胞与期望的基因改造的研究。我们已经使用 CRISPR/Cas9 技术的修改干细胞, 这是现在被用于广泛的应用, 包括敲门和敲除, large-scale 基因组删除, 汇集图书馆筛选的基因发现, 基因工程许多模型有机体和基因疗法9,10,11。这项技术涉及形成的配合物的链球菌化脓衍生 Cas9 核酸和 20-滨海指南 rna, 实现目标识别通过基地配对与基因组目标序列相邻的 3 ‘ 核苷酸 protospacer 相邻主题 (PAM) 序列。Cas9 核酸诱发双链断裂〜3核苷酸从 PAM 网站, 这是随后修复主要由同源结束连接 (NHEJ) 通路导致插入或删除在开放阅读框架, 从而功能基因敲除12。
我们改进的协议包括了人类胰腺细胞培养的细节, 对分裂灭活小鼠胚胎成纤维细胞 (MEFs) 的重新编程, 以达到更高的重编程效率, 随后适应无饲养的文化在基质, 定性为建立的干细胞, CRISPR 引导 RNA 设计和准备, 交付到干细胞作为 RNP 复合体, 单细胞排序生成克隆线编辑干细胞, 容易筛选和识别编辑, 并表征单细胞克隆。本研究通过引入 Cas9 蛋白和两个 CRISPR sgRNA RNP 配合物来诱导双链断裂 (DSBs) 并删除干预段, 从而有效地生成了基因组缺失。这种方法利用了两个指南, 在开放阅读框架中产生删除, 高效率的 NHEJ 导致低数量的克隆需要的特点, 并容易初步筛选克隆的自动毛细管电泳装置, 碎片分析仪。这些有效的基因组编辑方法, 以生成人类干细胞疾病模型将很快成为一个标准和常规的方法在任何干细胞实验室。最后, 精确的基因组编辑将使其超越干细胞疾病模型, 并有可能帮助催化细胞疗法。
Protocol
Representative Results
Discussion
重组人体体细胞干细胞, 为基础生物学研究、个性化医学、疾病建模、药物开发和再生医学等领域提供了重要的推动力16。许多当前和广泛使用的人类 iPSC 世代的方法要求使用病毒与集成入宿主基因组或 episomal 载体的风险, 以低重编程效率。在这里, 我们提出了一种有效的方法, 产生无饲养干细胞的人胰腺细胞与仙台病毒, 这导致 “无足迹” 和有效的重新编程。由此产生的人类干细?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
实验室的工作得到了博士后奖学金的资助, Dr. 安贾莉 Nandal, 以及马里兰州干细胞研究基金对 BT (TEDCO) 的探索性赠款。
Materials
| Sendai viral vectors – CytoTune-iPS 2.0 Kit | Invitrogen | A16517 | Thaw on ice; S No: 1 |
| Trypsin EDTA | Gibco Life Tech | 25300-054 | 0.05%, 100 ml; S No: 2 |
| Rock inhibitor (Y-27632) | Milipore | SCM075 | Use 10 μM; S No: 3 |
| DMEM/F-12 medium | Invitrogen | 11330-032 | S No: 4 |
| Serum replacement (KSR) | Gibco | 10828028 | S No: 5 |
| DMEM | Invitrogen | 11960069 | 1X; S No: 6 |
| Fetal bovine serum | Thermo Scientific | SH30071.03 | Aliquot; S No: 7 |
| L-glutamine (Glutamax, 100X), liquid | Thermo Scientific | 35050061 | 1/100; S No: 8 |
| Non-Essential Amino Acids | Gibco | 11140-050 | 1/100; S No: 9 |
| 2-Mercaptoethanol | Gibco | 21985023 | 55 mM, 1/1,000; S No: 10 |
| Hausser Hemacytometers | Hausser Scientific | 02-671-54 | S No: 11 |
| 0.1% Gelatin Solution | STEMCELL Technologies | 7903 | Incubate at 37º C for 1 hour; S No: 12 |
| SSEA-4 antibody | Santacruz | sc-21704 | 1/100; S No: 13 |
| TRA-1-81 antibody | Cell Signaling | 4745S | 1/200; S No: 14 |
| OCT4 antibody | Santa Cruz | sc-5279 | 1/1,000; S No: 15 |
| Collagen I, Rat Tail | Life Technologies | A10483-01 | Keep cold; S No: 16 |
| Alexa Fluor fluorescent 488/ 568 (secondary antibodies) | Invitrogen | A21202/A10042 | 1/2,000; S No: 17 |
| DPBS | Hyclone | SH30028LS | 1X; S No: 18 |
| 100-mm tissue culture dish | Falcon | 353003 | S No: 20 |
| 96-well tissue culture plate | Falcon | 353078 | S No: 21 |
| 6-well tissue culture plate | Falcon | 353046 | S No: 22 |
| Dissecting scope | Nikon | SMZ745 | S No: 23 |
| Picking hood | NuAire | NU-301 | S No: 24 |
| 15 ml Centrifuge Tube | Greiner Bio-One | 188271 | S No: 25 |
| 50 ml Centrifuge Tube | Greiner Bio-One | 227261 | S No: 26 |
| Sodium pyruvate | Invitrogen | 11360 | S No: 28 |
| β-mercaptoethanol | Sigma | M7522 | S No: 29 |
| Prigrow III medium | ABM | TM003 | S No: 31 |
| Countess™ Cell Counter | Invitrogen | C10227 | S No: 32 |
| Faxitron X-ray system | Faxitron | CellRad | S No: 33 |
| Accutase | Innovative cell Technologies | AT-104 | S No: 34 |
| Collagenase | Life Technologies | 17104019 | 1mg/ml stock; S No: 35 |
| Dispase | STEMCELL Technologies | 7923 | S No: 36 |
| hESC qualified matrigel | BD Biosciences | 354277 | To dilute, use cold DMEM/F-12; S No: 37 |
| bFGF | R & D | 233-FB | Stock 10 ug/ml; S No: 38 |
| Paraformaldehyde | EMS | 15710 | 4% stock in PBS; S No: 39 |
| TRA-1-60 | Santa Cruz | sc-21705 | 1/100; S No: 40 |
| NANOG | ReproCELL | RCAB0004P-F | 1/100; S No: 41 |
| Tween 20 | Sigma | P9416-100ML | S No: 42 |
| Alkaline Phosphatase kit | Stemgent | 00-0055 | S No: 43 |
| Cas9 protein | PNA Bio | CP01-50 | Thaw and aliquot; S No: 44 |
| Goat or donkey serum | Sigma | D9663/G9023 | S No: 45 |
| Triton X-100 | Sigma | X100-100ML | S No: 46 |
| DAPI | Thermo Scientific | D1306 | S No: 47 |
| Tris | Sigma | 9285-100ML | S No: 48 |
| NaCL | Sigma | S7653-250G | S No: 49 |
| EDTA | Sigma | BP2482-500 | S No: 50 |
| T4 DNA ligase | NEB | M0202T | S No: 51 |
| Mega Shortscript T7 kit | Thermo Scientific | AM1354 | S No: 52 |
| Mega Clear kit | Thermo Scientific | AM1908 | S No: 53 |
| SMC4 | BD Biosciences | 354357 | S No: 54 |
| Fibronectin | STEMCELL Technologies | 7159 | S No: 55 |
| CloneJET cloning kit | Thermo Scientific | K1232 | S No: 56 |
| Fragment analyzerTM | Advanced Analytical | S No: 57 | |
| mTeSR1 medium kit | STEMCELL Technologies | 5850 | Warm to room temperature; S No: 58 |
| Freezing medium mFreSR™ | STEMCELL Technologies | 5855 | S No: 59 |
| Freezing medium CryoStor® | STEMCELL Technologies | 7930 | S No: 60 |
| MEFs | Globalstem | GSC-6301G | S No: 61 |
| L-glutamine | Invitrogen | 25030081 | S No: 62 |
| Human pancreatic cells | ABM | T0159 | S No: 63 |
| STEMdiff™ Neural Induction Medium | Stemcell Technologies | 5835 | S No: 64 |
| RPMI | Thermofisher | 11875-093 | S No: 65 |
| 2% B27-insulin | Thermofisher | A1895601 | S No: 66 |
| CHIR99021 | Stemcell Technologies | 72052 | S No: 67 |
| IWP4 | Stemcell Technologies | 72552 | S No: 68 |
| 2% B27 | Thermofisher | 17504044 | S No: 69 |
| MCDB 131 | Life Technologies | 10372019 | S No: 70 |
| Sodium bicarbonate | Sigma-Aldrich | S8761-100ML | S No: 71 |
| Glucose | Sigma-Aldrich | G8270-100G | S No: 72 |
| BSA | Proliant | 68700 | S No: 73 |
| GDF8 | Pepro-Tech | 120-00 | S No: 74 |
| TUJ1 antibody | EMD Milipore | AB9354 | S No: 75 |
| NKX2-5 antibody | Santa Cruz | Sc-14033 | S No: 76 |
| SOX17 antibody | R & D systems | AF1924 | S No: 77 |
| Propidium iodide | Thermo Scientific | P3566 | S No: 78 |
| Amaxa 4D-nucleofector™ | Lonza | AAF-1002 | S No: 79 |
| FACSAria II (cell sorter) | BD biosciences | SORP UV | S No: 80 |
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