电穿孔介导的Cas9核糖核蛋白和mRNA递送到新鲜分离的初级小鼠肝细胞中
Summary
该协议描述了从肝脏中分离原代小鼠肝细胞并将CRISPR-Cas9电冲为核糖核蛋白和mRNA的技术，以破坏与肝脏遗传性代谢疾病相关的治疗靶基因。所描述的方法导致电穿孔后的高生存能力和高水平的基因修饰。
Abstract
该协议描述了一种快速有效的方法，用于分离原代小鼠肝细胞，然后电穿孔介导的CRISPR-Cas9作为核糖核蛋白（RNP）和mRNA递送。使用三步逆行灌注方法分离原代小鼠肝细胞，结果高产率高达50×每肝106 个细胞，细胞活力>85%。该方案提供了肝细胞电镀、染色和培养的详细说明。结果表明，电穿孔提供了89%的高转染效率，这是通过小鼠肝细胞中绿色荧光蛋白（GFP）阳性细胞的百分比和>35%的适度细胞活力来测量的。
为了证明这种方法的实用性，将靶向羟基苯基丙酮酸二加氧酶基因的CRISPR-Cas9电穿孔到初级小鼠肝细胞中作为原理验证基因编辑，以破坏与肝脏遗传代谢疾病（IMD）相关的治疗基因。RNP的靶向编辑率更高，为78%，而mRNA的编辑效率为47%。使用白蛋白测定 法在体外 评估肝细胞的功能，该测定表明将CRISPR-Cas9作为RNP和mRNA递送导致原代小鼠肝细胞中具有相当的细胞活力。该协议的一个有希望的应用是为影响肝脏的人类遗传疾病生成小鼠模型。
Introduction
肝脏IMD是遗传性疾病，其特征在于缺乏参与代谢的关键肝酶，导致有毒代谢物的积累。如果不进行治疗，肝脏IMD会导致器官衰竭或过早死亡1，2。肝脏IMD患者的唯一治疗选择是原位肝移植，由于供体器官的可用性低以及手术后免疫抑制治疗的并发症，这是有限的3，4。根据器官采购和移植网络最近收集的数据，肝移植等待名单上只有40%-46%的成年患者接受器官，而这些患者中有12.3%在等待名单上死亡5。此外，只有5%的罕见肝病有FDA批准的治疗方法6。很明显，迫切需要对肝脏IMDs进行新的治疗。然而，需要适当的疾病模型来开发新的治疗方案。
使用 体外 和 体内 系统对人类疾病进行建模仍然是开发有效疗法和研究肝脏IMD病理学的障碍。来自罕见肝病患者的肝细胞很难获得7。动物模型对于发展对疾病病理学的理解和测试治疗策略至关重要。然而，一个障碍是从携带致命突变的胚胎中生成模型。例如，试图创建Alagille综合征（ALGS）的小鼠模型，其中胚胎含有 Jag1 基因5′端附近5 kb序列的纯合子缺失，导致胚胎8的早期死亡。此外，通过在胚胎干细胞中进行基因编辑来生成小鼠模型可能是时间和资源密集型的9。最后，突变将出现在目标组织之外，导致混淆变量，可能阻碍对疾病的研究9。体细胞基因编辑将允许在肝脏组织中更容易地进行编辑，并绕过与使用胚胎干细胞生成模型相关的挑战。
电穿孔使CRISPR-Cas9能够通过施加高压电流使细胞膜透化而直接输送到细胞核中，并且与许多细胞类型兼容，包括那些对转染技术不妥协的细胞类型，例如人胚胎干细胞，多能干细胞和神经元10，11，12.然而，低生存能力是电穿孔的潜在缺点;优化程序可以产生高水平的递送，同时限制毒性13。最近的一项研究表明，将CRISPR-Cas9组分电穿孔到初级小鼠和人肝细胞中作为一种高效的方法的可行性14.肝细胞中的 离体 电穿孔有可能应用于为肝脏的人类IMD生成新的小鼠模型。
该协议提供了详细的分步程序，用于从肝脏中分离小鼠肝细胞，随后电穿孔CRISPR-Cas9作为RNP复合物，由Cas9蛋白和合成单向导RNA（sgRNA）组成，或Cas9 mRNA与sgRNA结合以获得高水平的靶向基因编辑。此外，该协议还提供了在CRISPR-Cas9电穿孔到新分离的小鼠肝细胞后量化基因编辑效率，活力和功能的方法。
Protocol
动物实验都是按照机构动物护理和使用委员会指南和克莱姆森大学批准的协议进行的。在8至10周龄的麻醉野生型C57BL / 6J小鼠中进行外科手术。 1. 动物手术 溶液和仪器的制备注意：灌注溶液和麻醉鸡尾酒配方显示在 材料表中。制备灌注液 1（肝素、EGTA 和 EBSS）、灌注液 2（聚乙二醇、乙二醇、高分子灌注酶、氯化钙2 和镁质<su…
Representative Results
从肝脏中分离出可平板的原代肝细胞肝脏灌注和肝细胞分离的整个过程如图 1所示。在该实验中，使用了野生型，8-10周龄的C57BL6 / 6J小鼠。该程序预计每只小鼠产生20-50个×106 个细胞，存活率在85%至95%之间。如果活力<70%，则应遵循全唇处理以去除死细胞。新鲜分离的肝细胞应接种在胶原 I 包被或含氮组织培养板上。在电镀的3-12小时内，预计肝细胞将?…
Discussion
肝细胞分离方案中概述的步骤具有挑战性，需要熟练练习。从肝脏中成功分离肝细胞有几个关键步骤。首先，下腔静脉的适当插管对于完全肝脏灌注至关重要。灌注后肝脏无变白表明导管移位（表1）。下腔静脉（逆行灌注）在手术中管，因为它比门静脉（顺行灌注）更简单，更容易获得17，18。一些方案使用缝合线来固定导管1…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
RNC获得了南卡罗来纳州生物工程再生和组织形成试点基金的资助，该基金由美国国立卫生研究院国家普通医学科学研究所（NIGMS），美国肝病研究基金会和美国基因&细胞治疗学会资助，拨款号为P30 GM131959，2021000920， 和2022000099分别。内容完全由作者负责，并不一定代表美国基因与细胞治疗学会或美国肝病研究协会基金会的官方观点。 图1 的原理图是用 BioRender.com 创建的。
Materials
| Equipment | |||
| 0.2 mL PCR 8-tube FLEX-FREE strip, attached clear flat caps, natural | USA Scientific | 1402-4700 | |
| 6-well Collagen Plates | Advanced Biomatrix | 5073 | |
| accuSpin Micro 17R | Fisher Scientific | 13-100-675 | |
| All-in-one Fluorescent Microscope | Keyence | BZ-X810 | |
| Analog Vortex Mixer | VWR | 97043-562 | |
| ART Wide BORE filtered tips 1,000 µL | ThermoFisher Scientific | 2079GPK | |
| Automated Cell Counter | Bio-Rad Laboratories | TC20 | |
| Blue Wax Dissection Tray | Braintree Scientific Inc. | DTW1 | 9" x 6.5" x 1/2"+F21 |
| Cell scraper/lifter | Argos Technologies | UX-04396-53 | Non-pyrogenic, sterile |
| Conical tubes (15 mL) | Fisher Scientific | 339650 | |
| Conical tubes (50 mL) | Fisher Scientific | 14-432-22 | |
| Cotton applicators | Fisher Scientific | 22-363-170 | |
| Curved scissors | Cooper Surgical | 62131 | |
| Disposable Petri Dishes | Falcon | 351029 | 100mm,sterile |
| Disposable Petri Dishes | VWR | 25373-100 | 35mm, sterile |
| Epoch Microplate Spectrophotometer | BioTek Instruments | 250082 | |
| Falcon Cell strainer (70 µm) | Fisher Scientific | 08-771-2 | |
| Forceps | Cooper Surgical | 61864 | Euro-Med Adson Tissue Forceps |
| IV catheters | BD | 382612 | 24 G x 0.75 in |
| IV infusion set | Baxter | 2C6401 | |
| MyFuge 12 Mini Centrifuge | Benchmark Scientific | 1220P38 | |
| Needles | Fisher Scientific | 05-561-20 | 25 G |
| Nucleofector 2b Device | Lonza | AAB-1001 | Program T-028 was used for electroporation in mouse hepatocytes |
| Peristaltic Pump | Masterflex | HV-77120-42 | 10 to 60 rpm; 90 to 260 VAC |
| Precision pump tubing | Masterflex | HV-96410-14 | 25 ft, silicone |
| Primaria Culture Plates | Corning Life Sciences | 353846 | Nitrogen-containing tissue culture plates |
| Serological Pipets (25 mL) | Fisher Scientific | 12-567-604 | |
| Syringes | BD | 329464 | 1 mL, sterile |
| T100 Thermal Cycler | Bio-Rad Laboratories | 1861096 | |
| Water bath | ALT | 27577 | Thermo Scientific Precision Microprocessor Controlled 280 Series, 2.5 L |
| Reagents | |||
| Alt-R S.p. Cas9 Nuclease V3 | IDT | 1081058 | |
| Beckman Coulter AMPure XP, 5 mL | Fisher Scientific | NC9959336 | |
| CleanCap Cas9 mRNA | Trilink Biotechnologies | L-7606-100 | |
| CleanCap EGFP mRNA | Trilink Biotechnologies | L-7201-100 | |
| Corning Matrigel Matrix | Corning Life Sciences | 356234 | |
| DMEM | ThermoFisher Scientific | 11885076 | Low glucose, pyruvate |
| Ethanol 70% | VWR | 71001-652 | |
| Fetal bovine serum | Thermoscientific | 26140-079 | |
| Hepatocyte Maintenance Medium (MM) | Lonza | MM250 | |
| Hepatocyte Plating Medium (PM) | Lonza | MP100 | |
| Mouse Albumin ELISA Kit | Fisher Scientific | NC0010653 | |
| Mouse/Rat Hepatocyte Nucleofector Kit | Lonza | VPL-1004 | |
| OneTaq HotStart DNA Polymerase | New England Biolabs | M0481L | |
| PBS 10x pH 7.4 | Thermoscientific | 70011-044 | No calcium or magnesium chloride |
| Percoll (PVP solution) | Santa Cruz Biotechnology | sc-500790A | |
| Periodic acid | Sigma-Aldrich | P7875-25G | |
| Permount Mounting Medium | VWR | 100496-550 | |
| QuickExtract DNA Extraction Solution | Lucigen Corporation | QE05090 | |
| Schiff’s fuchsin-sulfite reagent | Sigma-Aldrich | S5133 | |
| Trypsin-EDTA (0.25%) | ThermoFisher Scientific | 25200056 | Phenol red |
| Vybrant MTT Cell Viability Assay | ThermoFisher Scientific | V13154 | |
| Perfusion Solution 1 (pH 7.4, filter sterilized) | Stable at 4 °C for 2 months | ||
| EBSS | Fisher Scientific | 14155063 | Complete to 200 mL |
| EGTA (0.5 M) | Bioworld | 40520008-1 | 200 µL |
| HEPES (pH 7.3, 1 M) | ThermoFisher Scientific | AAJ16924AE | 2 mL |
| Perfusion Solution 2 (pH 7.4, filter sterilized) | Stable at 4 °C for 2 months | ||
| CaCl2·2H20 (1.8 mM) | Sigma | C7902-500G | 360 µL of 1 M stock |
| EBSS (no calcium, no magnesium, no phenol red) | Fisher Scientific | 14-155-063 | Complete to 200 mL |
| HEPES (1 M) | ThermoFisher Scientific | AAJ16924AE | 2 mL |
| MgSO4·7H20 (0.8 mM) | Sigma | 30391-25G | 160 µL of 1 M stock |
| Perfusion Solution 3 | Prepared fresh prior to use | ||
| Solution 2 | 50 mL | ||
| Liberase | Roche | 5401127001 | 0.094 Wunsch units/mL |
| Mouse Anesthetic Cocktail | |||
| Acepromzine | 0.25 mg/mL final concentration | ||
| Ketamine | 7.5 mg/mL final concentration | ||
| Xylazine | 1.5 mg/mL final concentration | ||
| Software | URL | ||
| Benchling | https://www.benchling.com/ | ||
| ImageJ | https://imagej.nih.gov/ij/ | ||
| TIDE: Tracking of Indels by Decomposition | https://tide.nki.nl/ |
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Diesen Artikel zitieren
Rathbone, T., Ates, I., Stuart, C., Parker, T., Cottle, R. N. Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes. J. Vis. Exp. (184), e63828, doi:10.3791/63828 (2022).