一种基于报告基因的细胞检测，用于监测剪接效率
Summary
该协议描述了一种微型基因报告基因检测，用于监测5′-剪接位点突变对剪接的影响，并开发抑制因子U1 snRNA以挽救突变诱导的剪接抑制。报告基因和抑制子U1 snRNA构建体在HeLa细胞中表达，并通过引物延伸或RT-PCR分析剪接。
Abstract
在基因表达过程中，前mRNA剪接的关键步骤涉及精确识别剪接位点和高效组装剪接体复合物，以在成熟mRNA的细胞质输出之前加入外显子并去除内含子。剪接效率可以通过剪接位点突变的存在，反式剪接因子的影响或治疗活性来改变。在这里，我们描述了可用于监测任何给定外显子的剪接效率的细胞测定方案。该测定使用适应性强的质粒编码为3-外显子/2-内含子的微型基因报告基因，可通过瞬时转染在哺乳动物细胞中表达。转染后，分离总细胞RNA，并通过引物延伸或半定量逆转录酶 – 聚合酶链反应（RT-PCR）确定报告mRNA中外显子剪接的效率。我们描述了如何通过在报告中引入疾病相关的5′剪接位点突变来确定其影响;以及如何通过与U1小核RNA（snRNA）构建体共转染来实现这些突变的抑制，该构建体在其5’区域中携带代偿性突变，该区域与前mRNA中外显子 – 内含子连接处的5′-剪接位点基本配对。因此，报告基因可用于设计治疗性U1颗粒，以提高对突变体5′剪接位点的识别。将 顺式作用调节位点（例如剪接增强剂或消音器序列）插入报告程序中也可用于检查U1 snRNP在由特定替代剪接因子介导的调节中的作用。最后，将报告细胞与小分子孵育，以确定潜在疗法对组成前mRNA剪接或携带突变5′剪接位点的外显子的影响。总体而言，报告基因检测可用于监测各种条件下的剪接效率，以研究基本的剪接机制和剪接相关疾病。
Introduction
前mRNA剪接是一个重要的处理步骤，它去除非编码内含子并精确地标记编码外显子以形成成熟的mRNA。通过剪接机械的组件识别外显子-内含子交界处的共识序列，称为5′-拼接位点和3‘-拼接位点，启动拼接过程。U1小核核糖核蛋白（snRNP）通过将U1 snRNA与前mRNA1的碱基配对来识别5‘-剪接位点。改变5‘-剪接位点序列的遗传突变与许多疾病有关2，3。据预测，U1 snRNA与突变5‘-剪接位点的碱基配对的损失会导致异常剪接，这可能会损害受影响转录本的翻译。纠正剪接缺陷的潜在治疗方法包括通过修饰的U1 snRNA在其5‘-区域携带代偿核苷酸变化来抑制突变，该区域与5‘-剪接位点基本配对。这种修饰的U1 snRNA，也称为外显子特异性U1 snRNA，已被发现可有效逆转剪接缺陷，导致获救的mRNA4，5，6，7，8的蛋白质表达增加。
在这里，我们描述了U1 snRNP补集测定，这是一种基于报告的细胞剪接测定，可以评估5‘-ss突变对外显子剪接的影响，也可用于开发修饰的U1 snRNA，以挽救外显子包涵体。我们还提供通过引物延伸和RT-PCR监测剪接报告转录本的方案，以及通过引物延伸和RT-qPCR确定修饰的U1 snRNA的表达的方案。
Protocol
1. 试剂和缓冲液 注意：所有使用真空过滤器的灭菌都应在生物安全柜中使用0.2μm聚醚砜（PES）膜进行。 将1.0 mL焦碳酸二乙酯（DEPC）加入1.0升去离子水中，在室温（RT）下混合至少1小时，高压灭菌两次，然后在使用前冷却至室温，以制备不含RNase的水。 通过将一包DMEM粉末（13.4克），3.7克碳酸氢钠，100毫升胎牛血清（FBS），青霉素和链霉素与约800毫升无菌去离?…
Representative Results
剪接报告人Dup51是一种三外显子二内含子微型基因，来源于人类β珠蛋白基因，之前已有描述（图1A）11，12 。我们通过引入Usher综合征相关的5’剪接位点突变创建了一个突变报告者Dup51p，这些突变发生在原钙粘蛋白15（PCDH15）基因13的外显子3中。外显子2-内含子2结处的5′-接头位点序列从CAG/GUUGGUAUC改为AUG/GUGUGUA…
Discussion
该测定可以适用于HeLa以外的细胞系中的剪接分析，但是，可能需要优化影响转染效率的因素，例如细胞汇合度和DNA数量。报告者与U1构建比是另一个关键参数，可能需要根据在其他细胞类型中观察到的表达水平来确定。提取的RNA的质量对于剪接分析至关重要;因此，强烈建议使用不含RNase的水并用RNase灭活剂对表面进行去污。
通过引物延伸或荧光RT-PCR对报告转录本的分析产生类…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院（R21CA170786和R01GM127464）和美国癌症协会（机构研究资助74-001-34-IRG）以及山谷研究合作计划（P1-4009和VRP77）向S.S.和W.M的资金支持。内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。
Materials
| Reagent Grade Deionized Water | ThermoFisher Scientific | 23-751628 | |
| Diethyl pyrocarbonate (DEPC) | Sigma-Aldrich | D5758-25ML | |
| Dulbecco's Modified Eagle Medium (DMEM) powder packet | Gibco | 12100-046 | |
| Sodium Bicarbonate | ThermoFisher Scientific | S233-500 | |
| Fetal Bovine Serum (FBS), Australian Source, Heat Inactivated | Omega Scientific | FB-22 | |
| Penicillin-Streptomycin (P/S) | Sigma-Aldrich | P4458-100ML | |
| Sodium Hydroxide, Standard Solution 1.0N | Sigma-Aldrich | S2567-16A | |
| Hydrochloric Acid, Certified ACS Plus, 36.5 to 38.0% | ThermoFisher Scientific | A144-500 | |
| Disposable PES Bottle Top Filters | ThermoFisher Scientific | FB12566510 | |
| EDTA Disodium Salt Dihydrate | Amresco | 0105-2.5KG | |
| 2.5% Trypsin (10x), no phenol red | ThermoFisher Scientific | 15090046 | |
| Sodium Chloride | Fisher Bioreagent | BP358-212 | |
| Potassium Chloride | Fisher Bioreagent | BP366-1 | |
| Disodium Hydrogen Phosphate Heptahydrate | Fisher Bioreagent | BP332-1 | |
| Potassium Dihydrogen Phosphate | Fisher Bioreagent | BP362-1 | |
| Transfection medium – Opti-MEM™ I Reduced Serum Medium, no phenol red | ThermoFisher Scientific | 11058021 | |
| Transfection Reagent – Lipofectamine™ 2000 | ThermoFisher Scientific | 13778150 | |
| TRIzol™ Reagent | ThermoFisher Scientific | 15596018 | |
| Chloroform (Approx. 0.75% Ethanol as Preservative/Molecular Biology) | ThermoFisher Scientific | BP1145-1 | |
| Ethanol, Absolute (200 Proof), Molecular Biology Grade, Fisher BioReagents | ThermoFisher Scientific | BP2818-4 | |
| Isopropanol, Molecular Biology Grade, Fisher BioReagents | ThermoFisher Scientific | BP2618-212 | |
| Glycogen (5 mg/ml) | ThermoFisher Scientific | AM9510 | |
| Direct-zol RNA Miniprep Kit | Zymo Research | R2052 | |
| ATP, [γ-32P]- 6000Ci/mmol 150mCi/ml Lead, 1 mCi | PerkinElmer | NEG035C001MC | |
| T4 Polynucleotide Kinase | New England Biolabs | M0201L | |
| Size exclusion beands – Sephadex® G-25 | Sigma-Aldrich | G2580-10G | |
| Size exclusion mini columns | USA Scientific | 1415-0600 | |
| pBR322 DNA-MspI Digest | New England Biolabs | N3032S | |
| Low Molecular Weight Marker, 10-100 nt | Affymetrix | 76410 100 UL | |
| Rnase inactivating reagents – RNaseZAP™ | Sigma-Aldrich | R2020-250ML | |
| dNTP Mix (10 mM ea) | ThermoFisher Scientific | 18427013 | |
| RNaseOUT™ Recombinant Ribonuclease Inhibitor | ThermoFisher Scientific | 10777019 | |
| Reverse Transcriptase – M-MLV Reverse Transcriptase | ThermoFisher Scientific | 28025013 | used for primer extension |
| Taq DNA Polymerase | ThermoFisher Scientific | 10342020 | |
| Random Hexamers (50 µM) | ThermoFisher Scientific | N8080127 | |
| Real time PCR mix – SYBR™ Select Master Mix | ThermoFisher Scientific | 4472903 | |
| SuperScript™ III Reverse Transcriptase | ThermoFisher Scientific | 18080093 | used for cDNA preparation |
| Dithiothreitol (DTT) | ThermoFisher Scientific | 18080093 | |
| 5X First-Strand Buffer | ThermoFisher Scientific | 18080093 | |
| Formamide (≥99.5%) | ThermoFisher Scientific | BP228-100 | Review Material Safety Data Sheets |
| Bromophenol Blue sodium salt | Sigma-Aldrich | 114405-5G | |
| Xylene Cyanol FF | Sigma-Aldrich | 2650-17-1 | |
| Tris Base (White Crystals or Crystalline Powder/Molecular Biology) | ThermoFisher Scientific | BP152-5 | |
| Boric Acid (Crystalline/Electrophoresis) | ThermoFisher Scientific | BP168-500 | |
| Acrylamide: Bis-Acrylamide 19:1 (40% Solution/Electrophoresis) | ThermoFisher Scientific | BP1406-1 | Review Material Safety Data Sheets |
| Urea (Colorless-to-White Crystals or Crystalline Powder/Mol. Biol.) | ThermoFisher Scientific | BP169-212 | |
| Ammonium peroxodisulphate (APS) ≥98%, Pro-Pure, Proteomics Grade | VWR | M133-25G | |
| Sigmacote | Sigma-Aldrich | SL2-100ML | |
| N,N,N',N'-Tetramethylethylenediamine (TEMED) ≥99%, Ultrapure | VWR | 0761-25ML | Review Material Safety Data Sheets |
| Adjustable Slab Gel Systems, Expedeon | VWR | ASG-400 | |
| Vertical Gel Wrap™ Glass Plate Sets, 16.5 x 14.5cm | VWR | NGP-125NR | |
| Vertical Gel Wrap™ Glass Plate Sets, 16.5 x 22.0cm | VWR | NGP-200NR | |
| Vertical Gel Wrap™ Glass Plate Sets, 16.5 x 38.7cm | VWR | NGP-400NR | |
| GE Storage Phosphor Screens | Sigma-Aldrich | GE28-9564 | |
| Typhoon™ FLA 7000 Biomolecular Imager | GE Healthcare | 28-9610-73 AB | |
| Beckman Coulter LS6500 Liquid Scintillation Counter | GMI | 8043-30-1194 | |
| C1000 Touch Thermal Cycler | ThermoFisher Scientific | ||
| QuantStudio 6 Flex Real-Time PCR Systems | ThermoFisher Scientific |
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Citer Cet Article
Wong, J., Martelly, W., Sharma, S. A Reporter Based Cellular Assay for Monitoring Splicing Efficiency. J. Vis. Exp. (175), e63014, doi:10.3791/63014 (2021).