在体外合成在人体细胞修饰蛋白表达的mRNA的感应
Summary
在这个视频文章中，我们描述了诱导细胞蛋白表达的体外合成修饰的mRNA。
Abstract
外源递送的期望的细胞编码合成的信使RNA（mRNA），用于诱导蛋白质合成的具有在再生医学，基础细胞生物学，治疗疾病，和细胞的重编程领域的巨大潜力。在这里，我们描述了一步步的协议，用于生成修改的表达与诱导蛋白表达的减少的免疫激活潜力和增加的稳定性，产生的mRNA的质量控制，细胞的转染mRNA和验证通过流式细胞术。达用的eGFP基因的单一转染后3天，在转染的HEK293细胞中产生绿色荧光蛋白。在这个视频文章中，绿色荧光蛋白的mRNA的合成被描述为一个例子。但是，该过程可以适用于生产其它所需的基因。采用改良的mRNA的合成，细胞可被诱导以瞬时表达所需的蛋白，它们通常不会表达。
Introduction
在细胞中，转录出信使RNA（mRNA）的与mRNA的下面翻译成所需蛋白质确保细胞的正常功能。遗传性或获得性遗传性疾病可导致合成蛋白质的不足，功能失调而引起严重的疾病。因此，一种新的治疗方法来诱导产生缺失或有缺陷的蛋白质是外源递送合成的修饰基因导入细胞，对于所期望的蛋白质编码。因此，细胞触发合成功能蛋白，它们通常不能生产或自然不会需要。使用这种方法，遗传性疾病可以通过引入基因的被纠正，对于有缺陷或缺失的蛋白1码。 mRNA的治疗也可以用于疫苗接种到synthetize蛋白质抗原，其是由肿瘤细胞或病原体表达的。从而，宿主的免疫系统可被激活，有效地消除肿瘤细胞或防止INFEctions 2,3。此外，近年来，基因被成功地用于产生诱导的多能干细胞（iPS细胞）。为了这个目的，成纤维细胞，转染的mRNA诱导重编程的表达因子4-6，并将其与在再生医学中的巨大潜力转化中的iPSC。
先前，使用常规的mRNA的低稳定性和强免疫原性有关。因此，常规的mRNA的临床应用受到限制。然而，胞苷和尿苷的取代5-甲基胞苷和假尿嘧啶mRNA分子通过Kariko和同事内呈现的mRNA分子稳定的生物流体中，并极大地降低了免疫激活7-10，它现在允许修饰mRNA的临床适用性。
使用合成产生的改性的mRNA，所需基因转录物可以暂时在体内 11交付或体外诱导蛋白表达。所引入的基因是由蜂窝翻译机制在生理条件下转化。由于缺乏整合到宿主细胞基因组相比，病毒的基因治疗载体，肿瘤发生的风险，防止12,13。因此，使用改性合成基因疗法将会得到更好的临床认可的未来。
在这里，我们描述了用于生产改性的mRNA（ 图1），转染细胞的mRNA和蛋白表达的转染细胞中的评价的详细协议。
Protocol
1.增强质粒含有所需的编码DNA序列（CDS） 预暖的SOC培养基，其中包括在转化试剂盒，室温并含有100μg/ ml氨苄青霉素至37℃的LB琼脂平板上。平衡的水浴中以42℃。 解冻的化学组分E的一个小瓶大肠杆菌在冰上。 添加1-5微升质粒（第10至100毫微克）含有CDS成化学成分E.小瓶大肠杆菌 ，轻轻混匀。不要吹打混合细胞。加入质粒后，通过敲击管轻轻拌匀。 <…
Representative Results
使用含有的eGFP的CDS的一个的pCDNA 3.3质粒的修饰的eGFP基因的合成成立（ 图1）。通过PCR扩增插入物和聚T形拖尾之后，以大约1100碱基对长度的清晰条带被检测到（ 图2）。增加了IVT时间增加的mRNA（ 图3）的产量。 IVT之后，一个清晰的mRNA条带大约1100碱基对的长度被检测，对应于绿色荧光蛋白的mRNA的要生产的长度（ 图4）。 所产?…
Discussion
mRNA的疗法在再生医学，治疗的疾病和免疫接种领域的巨大潜力。在这段视频中，我们展示了生产稳定，修饰诱导的细胞蛋白表达的mRNA。使用该协议，其他期望的mRNA可被生成。 在体外合成修饰mRNA的允许细胞的转染具有所需的mRNA诱导靶蛋白的表达。由此，所需的蛋白瞬时表达的生理条件下，直到外源递送的mRNA被完全降解。
在此视频中，绿色荧光蛋白的表达，证实了3?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
该项目是在巴登 – 符腾堡州，德国资助的欧洲社会基金。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Cell culture | |||
| DMEM, high glucose | PAA | E15-009 | |
| FBS | Life Technologies | 10500 | |
| Penicillin/Streptomycin | PAA | P11-010 | 100x |
| L-glutamine | PAA | M11-004 | 200 mM |
| DPBS without calcium and magnesium | PAA | E15-002 | |
| 0.04% Trypsin / 0.03% EDTA | Promocell | C-41020 | |
| TNS | Promocell | C-41120 | Trypsin Neutralizing Solution, 0.05% trypsin inhibitor in 0.1% BSA |
| HEK-293 cells | ATCC | CRL-1573 | |
| Consumables | |||
| Tissue culture plates, 12-well | Corning | 3512 | |
| Cell culture flask (75 cm2) | Corning | 430641 | |
| DNase-and RNase-free 1.5 ml sterile microcentrifuge tubes | Eppendorf | 0030 121.589 | Safe-Lock, Biopur |
| 15 ml conical tubes | greiner bio-one | 188271 | |
| PCR clean and sterile epT.I.P.S. dualfilter pipette tips | Eppendorf | 10 µl: 022491202, 100 µl: 022491237, 1000 µl: 022491253 | |
| Cryovial | greiner bio-one | 122279-128 | Cryo.s |
| 14 ml polypropylene round bottom tube for bacterial culture | BD Falcon | 352059 | |
| Plasmid amplification and purification | |||
| pcDNA 3.3_eGFP Plasmid | Addgene | 26822 | |
| One Shot Top10 chemically component Escherichia coli | Invitrogen | C4040-10 | |
| Sterile water (Ampuwa) | Fresenius Kabi | 1636071 | |
| LB medium (Luria/Miller) | Carl Roth | X968.1 | Dissolve 25 g l-1 in sterile water. |
| LB agar (Luria/Miller) | Carl Roth | X969.1 | Dissolve 40 g l-1 in sterile water. |
| Ampicillin Ready Made Solution | Sigma Aldrich | A5354 | 100 mg/ml |
| Glycerol | Sigma Aldrich | G2025 | |
| QIAprep Spin Miniprep Kit | Qiagen | 27104 | |
| mRNA production | |||
| HotStar HiFidelity Polymerase Kit | Qiagen | 202602 | |
| QIAquick PCR Purification Kit | Qiagen | 28106 | |
| MEGAscript T7 Kit | Life Technologies | AM1334 | |
| 5-Methylcytidine-5´-triphosphate | Trilink | N1014 | 5-Methyl-CTP |
| Pseudouridine-5´-triphosphate | Trilink | N1019 | Pseudo-UTP |
| 3´-O-Me-m7G(5´)ppp(5`)G RNA cap structure analog | New England Biolabs | S1411L | |
| RiboLock RNase Inhibitor | Thermo Scientific | EO0381 | 40 U/µl |
| TURBO DNase | Life Technologies | AM1334 | 2 U/µl (from MEGAscript T7 Kit) |
| Antarctic phosphatase | New England Biolabs | MO289S | |
| RNeasy mini kit | Qiagen | 74104 | |
| RNaseZap solution | Life Technologies | AM9780 | |
| Transfection | |||
| Lipofectamine 2000 Transfection Reagent | Invitrogen | 11668-019 | Cationic lipid transfection reagent |
| Opti-MEM I Reduced Serum Media | Invitrogen | 11058-021 | Improved Minimal Essential Medium (MEM) with reduced Fetal Bovine Serum (FBS) supplementation |
| Gel electrophoresis | |||
| Agarose | Sigma-Aldrich | A9539 | |
| Gelred Nucleic Acid Gel Stain | Biotium | 41003 | 10,000x in water |
| peqGOLD Range Mix DNA-Ladder | Peqlab | 25-2210 | |
| 0.5-10 kb RNA Ladder | Invitrogen | 15623-200 | |
| 1x TBE buffer | |||
| Flow cytometry analyses | |||
| CellFIX (1x) | BD Biosciences | 340181 | 10x concentrate |
| Primer for insert amplification and poly (T) tail PCR | |||
| Forward Primer (HPLC-grade) 10 µM | Ella Biotech | 5´-TTGGACCCTCGTACAGAAGCTAATACG-3´ | |
| Reverse Primer (HPLC-grade) 10 µM | Ella Biotech | 5´- T120-CTTCCTACTCAGGCTTTATTCAAAGACCA-3´ | |
| Equipment | |||
| Cell incubator | Binder | CO2 (5%) and O2 (20%) | |
| CASY cell counter | Schärfe System | ||
| Sterile workbench | BDK Luft-und Reinraumtecknik GmbH | ||
| Bacterial incubator | Incutec | ||
| Water bath | |||
| ScanDrop spectrophotometer | Analytic Jena | ||
| PCR thermocycler | Eppendorf | ||
| Microcentrifuge | Eppendorf | ||
| Vortex | peqlab | ||
| Thermomixer | Eppendorf | ||
| Gel apparatus for electrophoresis | Bio-Rad | ||
| Gel documentation system | Bio-Rad | ||
| FACScan System | BD Biosciences | ||
| Fluorescence microscope | Nikon | ||
| Phase-contrast microscope | Zeiss |
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Cite This Article
Avci-Adali, M., Behring, A., Steinle, H., Keller, T., Krajeweski, S., Schlensak, C., Wendel, H. P. In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells. J. Vis. Exp. (93), e51943, doi:10.3791/51943 (2014).