CRISPR 介导的小脑颗粒细胞在宫内电穿孔基因转移中的功能分析损失
Summary
传统的功能丧失基因的研究使用挖空动物往往是昂贵和耗时。基于电穿孔的 CRISPR 介导体细胞诱变是了解体内基因功能的有力工具。在这里, 我们报告了一个方法来分析的淘汰赛表型的小脑增殖细胞。
Abstract
大脑畸形通常是由基因突变引起的。破译患者衍生组织中的突变, 确定了疾病的潜在致病因素。为了验证突变基因功能失调对疾病发展的贡献, 携带突变的动物模型的生成是一个明显的方法。虽然生殖基因工程小鼠模型 (GEMMs) 是流行的生物工具和展示重现性的结果, 它受到时间和成本的限制。同时, 非生殖 GEMMs 往往能更可行地探索基因功能。由于某些脑部疾病 (如脑肿瘤) 似乎是由躯体而不是生殖突变引起的, 非生殖嵌合体小鼠模型, 正常和异常细胞共存, 可能有助于疾病相关的分析。本研究报告了一种诱导小脑 CRISPR 介导的躯体突变的方法。具体地说, 我们利用条件敲鼠, 其中Cas9和GFP是长期激活的CAG (CMV 增强/鸡ß肌动蛋白) 启动后的基因组重组。自行设计的单导 rna (sgRNAs) 和 recombinase 序列, 都编码在一个单一质粒结构, 被交付到小脑茎/祖细胞在胚胎阶段使用的子宫电穿孔。因此, 转染细胞及其子细胞被标记为绿色荧光蛋白 (GFP), 从而促进进一步的表型分析。因此, 该方法不仅显示了以电穿孔为基础的基因传递到胚胎小脑细胞, 而且还提出了一种新的定量方法来评估 CRISPR 介导的丧失功能表型。
Introduction
脑部疾病是最可怕的致命疾病之一。它们通常是由基因突变和随后的失调引起的。为了了解脑部疾病的分子机制, 不断努力破译人类的基因组, 发现了一些潜在的致病基因。到目前为止, 生殖基因工程动物模型已被用于体内增益功能 (GOF) 和丧失功能 (LOF) 分析这些候选基因。由于功能验证研究的加速发展, 一种更可行、更灵活的体内基因检测系统对基因功能的研究是可取的。
应用体内电穿孔基因转移系统对小鼠脑发育有较好的作用。事实上, 在子宫电穿孔中使用的几项研究显示了它们在发育中的大脑1、2、3进行功能分析的潜力。实际上, 老鼠大脑的几个区域, 如大脑皮层4, 视网膜5, 间脑6, 后脑7, 小脑8, 脊髓9已被体细胞基因交付的目标方法, 到目前为止。
事实上, 胚胎小鼠脑内电穿孔的瞬态基因表达长期以来一直被用于 GOF 分析。最近基于座子的基因组集成技术进一步启用了10、11感兴趣基因的长期和/或有条件表达, 这有利于在空间和时间上解剖基因功能, 在发展。与 GOF 分析相比, LOF 分析更具挑战性。在对 siRNAs 和 shRNA 质粒进行瞬态转染的同时, 由于外部引入的核酸 (如质粒和 dsRNAs) 的最终降解, 不能保证基因 LOF 的长期影响。然而, CRISPR/Cas 技术在 LOF 分析中提供了突破。基因编码的荧光蛋白 (如GFP) 或生物发光蛋白 (如萤火虫荧光素酶) 已与 CRISPR-Cas9 和 sgRNAs, 以标签的细胞暴露在 CRISPR-Cas9-mediated 体细胞突变。然而, 这种方法在增殖细胞的功能研究中可能有局限性, 因为外源标记基因在长期增殖后会被稀释和降解。当转染细胞及其子细胞在基因组中经历 CRISPR 诱导的突变时, 它们的脚印可能会随着时间而消失。因此, 基因标记方法适合于解决这一问题。
我们最近开发了一种基于 CRISPR 的 LOF 方法在小脑颗粒细胞中经历长期增殖在他们的分化12。为了对转染细胞进行基因标记, 我们构建了一种携带 sgRNA 的质粒, 并将质粒引入 Rosa26-CAG-LSL-Cas9-P2A-EGFP 小鼠13 在宫内电穿孔中的小脑。与常规质粒载体编码 EGFP 不同, 这种方法成功地标记了转染的颗粒神经元前体 (大亚湾) 及其子细胞。该方法在正常脑发育和肿瘤易发背景下对增殖细胞感兴趣基因的体内功能有很大的支持作用。
Protocol
Representative Results
Discussion
使用外子宫内电穿孔, 我们以前报告 siRNA在体内功能分析的 Atoh1 在早期的小脑颗粒细胞分化8。由于 siRNA 稀释/降解和子宫壁外的胚胎暴露, 转颗粒细胞的表型分析仅限于胚胎阶段。然而, 目前的方法能够分析产后动物的表型。
我们以前的研究表明,在宫内电穿孔中抑癌Ptch1的 Cas9-mediated 敲除成功诱导髓母细胞2。相比…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢劳拉 Sieber, 安娜 Neuerburg, 亚辛哈琳, 和佩特拉 Schroeter 的技术援助。我们还感谢 Drs. k. Reifenberg、k. 戴尔和 Prückl 为 DKFZ 动物实验提供帮助;成像核心设施的 DKFZ 和卡尔蔡司成像中心在 DKFZ 的共焦显微成像。这项工作得到德意志 Forschungsgemeinschaft, KA 4472/1-1 (交流) 的支持。
Materials
| Alexa 488 Goat anti-Chicken | ThermoFisher | A11039 | 1:400 dilution |
| Alexa 568 Donkey anti-Mouse | Life Technologies | A-10037 | 1:400 dilution |
| Alexa 594 Donkey anti-Rabbit | ThermoFisher | A21207 | 1:400 dilution |
| Alexa 647 Donkey anti-Rabbit | Life Technologies | A31573 | 1:400 dilution |
| Alkaline Phosphatase (FastAP) | ThermoFisher | EF0654 | |
| Autoclave band | Kisker Biotech | 150262 | |
| BamHI (HF) | NEB | R3136S | |
| BbsI (FastDigest) | ThermoFisher | FD1014 | |
| Cellulose Filter Paper (Whatman) | Sigma-Aldrich | WHA10347525 | |
| Cloth | Tork | 530378 | |
| Confocal laser scanning microscope | Zeiss | LSM800 | |
| D-Luciferin | biovision | 7903-1 | |
| DAPI | Sigma-Aldrich | D9542 | 1:1000 dilution |
| Disposable plastic molds (Tissue-Tek Cyromold) | VWR | 4566 | |
| DMEM Glutamax | ThermoFisher | 31966047 | |
| Donkey serum | Sigma-Aldrich | D9663 | |
| EcoRI (HF) | NEB | R3101S | |
| Electro Square Porator | BTX | ECM830 | |
| Endofree Maxi Kit | Qiagen | 12362 | |
| Ethanol | Merck | 107017 | |
| Eye ointment (Bepanthen) | Bayer | 81552983 | |
| Fast Green | Merck | 104022 | |
| FBS | ThermoFisher | 10270-016 | |
| Filter (0.22 µm) | Merck | F8148 | |
| Fluorescent cell imager (ZOE) | Biorad | 1450031 | |
| Forceps straight | Fine Science Tools | 91150-20 | |
| Gauze (X100 ES-pads 8f 10 x 10 cm) | Fisher Scientific | 15387311 | |
| GFP antibody | Abcam | ab13970 | 1:1000 dilution |
| Gibson Assembly Master Mix | NEB | E2611S | |
| Glass Capillary with Filament | Narishige | GD1-2 | |
| Heating Pad | ThermoLux | 463265 / -67 | |
| Image Processing software (ImageJ and Fiji) | NIH | – | |
| Insulin syringe (B. Braun OMNICAN U-100) | Carl Roth | AKP0.1 | |
| Isoflurane | Zoetis | TU061219 | |
| IVIS Lumina LT Series III Caliper | Perkin Elmer | CLS136331 | |
| Kalt Suture Needles | Fine Science Tools | 12050-02 | |
| KAPA HIFI HOTSTART READY mix | Kapa Biosystems | KK2601 | |
| Ki67 antibody | Abcam | ab15580 | 1:500 dilution |
| Light Pointer | Photonic | PL3000 | |
| Liquid blocker pen | Kisker Biotech | MKP-1 | |
| Metamizol | WDT | – | |
| Microgrinder | Narishige | EG-45 | |
| Microinjector | Narishige | IM300 | |
| Micropipette Puller | Sutter Instrument Co. | P-97 | |
| Microscope software ZEN | Zeiss | – | |
| Non-sterile Silk Suture Thread (0.12 mm) | Fine Science Tools | 18020-50 | |
| O.C.T. Compound (Tissue-Tek) | VWR | 4583 | |
| p27 antibody | BD bioscience | 610241 | 1:200 dilution |
| Paraformaldehyde | Roth | 335.3 | |
| PBS (1x) | Life Technologies | 14190169 | |
| pCAG-EGxxFP | Addgene | 50716 | |
| Polyethylenimine | Sigma-Aldrich | 408727 | |
| pX330 plasmid | Addgene | 42230 | |
| QIAprep Spin Miniprep Kit | Qiagen | 27104 | |
| QIAquick Gel Extraction Kit | Qiagen | 28704 | |
| Quick Ligation Kit | NEB | M2200S | |
| Ring Forceps | Fine Science Tools | 11103-09 | |
| Slides (SuperFrost) | ThermoFisher | 10417002 | |
| Software for biostatistics (Prism 7) | GraphPad Software, Inc | – | |
| Spitacid | EcoLab | 3003840 | |
| Stereomicroscope | Nikon | C-PS | |
| Sucrose | Sigma-Aldrich | S5016 | |
| Surgical scissors | Fine Science Tools | 91460-11 | |
| Surgical scissors with blunt tip | Fine Science Tools | 14072-10 | |
| Suture (Supramid schwarz DS 16, 1.5 (4/0)) | SMI | 220340 | |
| T4 DNA Ligation Buffer | NEB | B0202S | |
| T4 PNK | NEB | M0201S | |
| Tissue scissors Blunt (11.5 cm) | Fine Science Tools | 14072-10 | |
| TOP2B antibody | Santa Cruz | sc13059 | 1:200 dilution |
| Trypsin (2.5 %) | ThermoFisher | 15090046 | |
| Tweezers w/5mm Ø disk electrodes Platinum | Xceltis GmbH | CUY650P5 | |
| Vaporizer | Drägerwerk AG | GS186 |
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