Name: efficient transfection of in vitro transcribed mrna in cultured cells using peptide-poloxamine nanoparticles
Uploaded: 2022-08-17T14:20:00
Description: Watch this Scientific Journal Video about Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles at JoVE.com

本研究由国家自然科学基金项目（国家自然科学基金委，批准号82041045和82173764）、国家科技部发病防疫技术体系研究重大专项（2021YFC2302500）、重庆市人才：优秀青年人才项目（CQYC202005027）和重庆市自然科学基金项目（cstc2021jcyj-msxmX0136）资助。作者感谢丁晓燕博士测量的流体动力直径（nm）和多分散指数（PDI）。

1. 化学修饰的mRNA的 体外 转录
注意：需要使用无核酸酶的试管，试剂，玻璃器皿，移液器吸头等，因为RNA酶在环境中无处不在，例如实验室溶液，仪器表面，头发，皮肤，灰尘等。使用前彻底清洁工作台表面和移液器，并戴上手套以避免RNase污染。
<ol><li>对 DNA 模板进行线性化。<ol><li>合成雌蕊花素酶（MetLuc）的开放阅读框（ORF），两侧是T7聚合酶启动子?...

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	<li>Nature milestones in vaccines. Springer Nature Available from: <a target="_blank" href="https://www.nature.com/collections/hcajdiajij">https://www.nature.com/collections/hcajdiajij</a> (2020)</li><li>Barbier, A. J., Jiang, A. Y., Zhang, P., Wooster, R., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+progress+of+mRNA+vaccines+and+immunotherapies.">The clinical progress of mRNA vaccines and immunotherapies.</a> Nature Biotechnology. 40 (6), 840-854 (2022).</li><li>Mulligan, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I/II+study+of+COVID-19+RNA+vaccine+BNT162b1+in+adults.">Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adults.</a> Nature. 586 (7830), 589-593 (2020).</li><li>Tang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnologies+in+delivery+of+DNA+and+mRNA+vaccines+to+the+nasal+and+pulmonary+mucosa.">Nanotechnologies in delivery of DNA and mRNA vaccines to the nasal and pulmonary mucosa.</a> Nanomaterials. 12 (2), 226 (2022).</li><li>Freyn, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multi-targeting,+nucleoside-modified+mRNA+influenza+virus+vaccine+provides+broad+protection+in+mice.">A multi-targeting, nucleoside-modified mRNA influenza virus vaccine provides broad protection in mice.</a> Molecular Therapy. 28 (7), 1569-1584 (2020).</li><li>Wu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+SARS-CoV-2+mRNA+vaccines+confer+broad+neutralization+as+primary+or+booster+series+in+mice.">Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice.</a> Vaccine. 39 (51), 7394-7400 (2021).</li><li>Chaudhary, N., Weissman, D., Whitehead, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+vaccines+for+infectious+diseases:+Principles,+delivery+and+clinical+translation.">mRNA vaccines for infectious diseases: Principles, delivery and clinical translation.</a> Nature Reviews Drug Discovery. 20, 817-838 (2021).</li><li>Kormann, M. S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+therapeutic+proteins+after+delivery+of+chemically+modified+mRNA+in+mice.">Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.</a> Nature Biotechnology. 29 (2), 154-157 (2011).</li><li>Tavernier, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+as+gene+therapeutic:+How+to+control+protein+expression.">mRNA as gene therapeutic: How to control protein expression.</a> Journal of Controlled Release. 150 (3), 238-247 (2011).</li><li>Walsh, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+immunogenicity+of+two+RNA-based+Covid-19+vaccine+candidates.">Safety and immunogenicity of two RNA-based Covid-19 vaccine candidates.</a> The New England Journal of Medicine. 383 (25), 2439-2450 (2020).</li><li>Baden, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+the+mRNA-1273+SARS-CoV-2+vaccine.">Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine.</a> The New England Journal of Medicine. 384 (5), 403-416 (2021).</li><li>Guan, S., Rosenecker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnologies+in+delivery+of+mRNA+therapeutics+using+nonviral+vector-based+delivery+systems.">Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems.</a> Gene Therapy. 24 (3), 133-143 (2017).</li><li>Sharova, L. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Database+for+mRNA+half-life+of+19+977+genes+obtained+by+DNA+microarray+analysis+of+pluripotent+and+differentiating+mouse+embryonic+stem+cells.">Database for mRNA half-life of 19 977 genes obtained by DNA microarray analysis of pluripotent and differentiating mouse embryonic stem cells.</a> DNA Research. 16 (1), 45-58 (2009).</li><li>Houseley, J., Tollervey, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+pathways+of+RNA+degradation.">The many pathways of RNA degradation.</a> Cell. 136 (4), 763-776 (2009).</li><li>Kowalski, P. S., Rudra, A., Miao, L., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivering+the+messenger:+Advances+in+technologies+for+therapeutic+mRNA+delivery.">Delivering the messenger: Advances in technologies for therapeutic mRNA delivery.</a> Molecular Therapy. 27 (4), 710-728 (2019).</li><li>Sahin, U., Karik&#243;, K., T&#252;reci, &. #. 2. 1. 4. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRNA-based+therapeutics-developing+a+new+class+of+drugs.">MRNA-based therapeutics-developing a new class of drugs.</a> Nature Reviews Drug Discovery. 13, 359-380 (2014).</li><li>Hajj, K. A., Whitehead, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+translation:+Non-viral+materials+for+therapeutic+mRNA+delivery.">Tools for translation: Non-viral materials for therapeutic mRNA delivery.</a> Nature Reviews Materials. 2, 17056 (2017).</li><li>Deering, R. P., Kommareddy, S., Ulmer, J. B., Brito, L. A., Geall, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+vaccines:+Prospects+for+non-viral+delivery+of+mRNA+vaccines.">Nucleic acid vaccines: Prospects for non-viral delivery of mRNA vaccines.</a> Expert Opinion on Drug Delivery. 11 (6), 885-899 (2014).</li><li>Wadhwa, A., Aljabbari, A., Lokras, A., Foged, C., Thakur, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+and+challenges+in+the+delivery+of+mRNA-based+vaccines.">Opportunities and challenges in the delivery of mRNA-based vaccines.</a> Pharmaceutics. 12 (2), 102 (2020).</li><li>Hou, X., Zaks, T., Langer, R., Dong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+nanoparticles+for+mRNA+delivery.">Lipid nanoparticles for mRNA delivery.</a> Nature Reviews Materials. 6 (12), 1078-1094 (2021).</li><li>Sahin, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COVID-19+vaccine+BNT162b1+elicits+human+antibody+and+T(H)1+T+cell+responses.">COVID-19 vaccine BNT162b1 elicits human antibody and T(H)1 T cell responses.</a> Nature. 586 (7830), 594-599 (2020).</li><li>Azzi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+immune+response+in+BNT162b2+COVID-19+vaccine+recipients.">Mucosal immune response in BNT162b2 COVID-19 vaccine recipients.</a> EBioMedicine. 75, 103788 (2022).</li><li>Guan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+peptide-poloxamine+nanoparticles+enable+in+vitro+and+in+vivo+genome+restoration+for+cystic+fibrosis.">Self-assembled peptide-poloxamine nanoparticles enable in vitro and in vivo genome restoration for cystic fibrosis.</a> Nature Nanotechnology. 14 (3), 287-297 (2019).</li><li>Pitard, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negatively+charged+self-assembling+DNA/poloxamine+nanospheres+for+in+vivo+gene+transfer.">Negatively charged self-assembling DNA/poloxamine nanospheres for in vivo gene transfer.</a> Nucleic Acids Research. 32 (20), 159 (2004).</li><li>Hiroi, T., Shibayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+particle+size+distribution+in+turbid+solutions+by+dynamic+light+scattering+microscopy.">Measurement of particle size distribution in turbid solutions by dynamic light scattering microscopy.</a> Journal of Visualized Experiments. (119), e54885 (2017).</li><li>Hassett, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+lipid+nanoparticle+size+on+mRNA+vaccine+immunogenicity.">Impact of lipid nanoparticle size on mRNA vaccine immunogenicity.</a> Journal of Controlled Release. 335, 237-246 (2021).</li><li>Suk, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+penetration+of+fresh+undiluted+sputum+expectorated+by+cystic+fibrosis+patients+by+non-adhesive+polymer+nanoparticles.">The penetration of fresh undiluted sputum expectorated by cystic fibrosis patients by non-adhesive polymer nanoparticles.</a> Biomaterials. 30 (13), 2591-2597 (2009).</li><li>Kim, N., Duncan, G. A., Hanes, J., Suk, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barriers+to+inhaled+gene+therapy+of+obstructive+lung+diseases:+A+review.">Barriers to inhaled gene therapy of obstructive lung diseases: A review.</a> Journal of Controlled Release. 240, 465-488 (2016).</li><li>Liu, Z., Fontana, F., Python, A., Hirvonen, J. T., Santos, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+production+of+particles:+Mechanism,+methodology,+and+applications.">Microfluidics for production of particles: Mechanism, methodology, and applications.</a> Small. 16 (9), 1904673 (2020).</li><li>Guan, S., Darmst&#228;dter, M., Xu, C., Rosenecker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+investigations+on+optimizing+and+nebulization+of+IVT-mRNA+formulations+for+potential+pulmonary-based+alpha-1-antitrypsin+deficiency+treatment.">In vitro investigations on optimizing and nebulization of IVT-mRNA formulations for potential pulmonary-based alpha-1-antitrypsin deficiency treatment.</a> Pharmaceutics. 13 (8), 1281 (2021).</li><li>Shepherd, S. J., Issadore, D., Mitchell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+formulation+of+nanoparticles+for+biomedical+applications.">Microfluidic formulation of nanoparticles for biomedical applications.</a> Biomaterials. 274, 120826 (2021).</li><li>Han, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+confined+impingement+jets+mixer+for+flash+nanoprecipitation.">A simple confined impingement jets mixer for flash nanoprecipitation.</a> Journal of Pharmaceutical Sciences. 101 (10), 4018-4023 (2012).</li></ol>

这里描述的方案不仅允许具有确定性质的IVT mRNA疫苗制剂的具有成本效益和快速生产，而且还提供了根据特定治疗目的（例如基因治疗）定制PP-sNp制剂的可能性。为了确保IVT mRNA/PP-sNp的成功生成，建议特别注意一些关键步骤。在使用mRNA时，请始终记住，应在整个过程中保持无RNase的条件，因为IVT mRNA对RNase的降解非常敏感，即使IVT mRNA是用化学修饰制备的。同时，在储存配方时，还必须确保无RNase的...

疫苗接种被誉为降低传染病发病率和死亡率的最有效医疗干预措施之一1.自2019年新型冠状病毒肺炎（COVID-19）爆发以来，疫苗的重要性已经得到证明。与注射灭活或减毒活病原体的传统概念相反，最先进的疫苗方法，如基于核酸的疫苗，侧重于保持目标病原体的免疫刺激特性，同时避免与传统的全微生物病毒或基于细菌的疫苗相关的潜在安全问题。基于DNA和RNA（即 体外 转录的信使RNA，IVT mRNA）的疫苗都表现出对多种疾病的预防治疗潜力，包括传染病和癌症2，3。原则上，基于核酸的疫苗的潜力与其生产、功效和安全性有关4.这些疫苗可以以无细胞的方式生产，以实现具有成本效益，可扩展和快速的生产。
基于单一核酸的疫苗可以编码多种抗原，从而能够以较少的接种次数靶向多种病毒变异或细菌，并增强对弹性病原体的免疫反应5，6。此外，基于核酸的疫苗可以模仿病毒或细菌感染的自然侵袭过程，带来B细胞和T细胞介导的免疫反应。与一些基于病毒或基于DNA的疫苗不同，基于IVT mRNA的疫苗在安全性方面具有巨大的优势。它们可以在细胞质基质中快速表达所需的抗原，并且不会整合到宿主基因组中，从而消除了对插入诱变7的担忧。IVT-mRNA在成功翻译后自动降解，因此其蛋白表达动力学可以很容易地控制在8，9。在严重急性呼吸综合征冠状病毒2（SARS-CoV-2）大流行的催化下，全球公司/机构的努力使许多类型的疫苗能够投放市场。基于IVT mRNA的疫苗技术显示出巨大的潜力，并且由于其快速设计和灵活的能力，可以在几个月内适应任何靶抗原，因此首次显示出其先前预期的成功。IVT mRNA疫苗抗击COVID-19的成功应用，不仅开启了IVT mRNA疫苗研发的新时代，也为快速开发应对传染病暴发的有效疫苗积累了宝贵经验10，11.
尽管IVT mRNA疫苗具有广阔的潜力，但IVT mRNA在细胞内有效递送到作用部位（即细胞质）仍然是一个主要障碍12，特别是对于 通过 气道4施用的疫苗。IVT mRNA本质上是一种不稳定的分子，具有极短的半衰期（~7小时）13，这使得IVT mRNA极易被无处不在的RNase14降解。在 体内 应用的情况下，先天免疫系统的淋巴细胞倾向于吞噬公认的IVT mRNA。此外，IVT mRNA的高负电荷密度和大分子量（1 x 104-10 10 6 Da）损害了其在细胞膜阴离子脂质双层中的有效渗透15。因此，需要具有某些生物官能材料的递送系统来抑制IVT mRNA分子的降解并促进细胞摄取16。
除了一些直接用于体内研究的裸IVT mRNA的特殊情况 外 ，还使用各种递送系统将IVT mRNA携带到作用17，18的治疗位点。先前的研究表明，在没有递送系统19的帮助下，在细胞质基质中仅检测到少数IVT mRNA。通过在该领域的不断努力，已经开发了许多策略来改善RNA递送，从鱼精蛋白缩合到脂质包封20。脂质纳米颗粒（LNP）是mRNA递送载体中临床上最先进的，所有经批准的临床用途mRNA COVID-19疫苗都采用基于LNP的递送系统21这一事实证明了这一点。然而，当制剂 通过 呼吸途径22递送时，LNPs不能介导有效的mRNA转染，这显着限制了这些制剂在诱导粘膜免疫应答或解决肺部相关疾病（如囊性纤维化或α1-抗胰蛋白酶缺乏症）中的应用。因此，需要开发一种新型递送系统，以促进气道相关细胞中IVT mRNA的高效递送和转染，以解决这一未满足的需求。
已经证实，肽-泊洛沙明自组装纳米颗粒（PP-sNp）递送系统可以介导小鼠呼吸道中核酸23的有效转染。PP-sNp采用多功能模块化设计方法，可以将不同的功能模块集成到纳米颗粒中，以便快速筛选和优化23。PP-sNp内的合成肽和电中性两亲性嵌段共聚物（泊洛沙明）可以与IVT mRNA自发相互作用，生成结构紧凑、表面光滑均匀分布的纳米颗粒23。PP-sNp可以提高IVT mRNA分子在培养细胞和小鼠呼吸道中的基因转染作用23。本研究描述了一种用于生成含有IVT mRNA的PP-sNp的方案，该MRNA编码甲基化物胺酶（甲基葡糖-mRNA）（图1）。该方案 采用 交错式人字形混合设计的微流体混合装置进行受控和快速混合。该过程易于执行，并允许生成尺寸更均匀的PP-sNp。使用微流体混合器生产PP-sNp的总体目标是以良好控制的方式产生用于mRNA络合的PP-sNp，从而实现高效且可重复 的体外细胞转染。本实验方案描述了含有甲基葡聚糖-mRNA的PP-sNp的制备、组装和表征。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BamHI</td>
			<td>Takara</td>
			<td>1010</td>
			<td></td>
		</tr>
		<tr>
			<td>cap 1 capping system</td>
			<td>Jinan</td>
			<td>M082</td>
			<td></td>
		</tr>
		<tr>
			<td>Dendritic cell-line</td>
			<td>Sigma</td>
			<td>SCC142</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA sequence</td>
			<td>Genescript</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human bronchial epithelial cells</td>
			<td>Sigma</td>
			<td>SCC150</td>
			<td></td>
		</tr>
		<tr>
			<td>KpnI</td>
			<td>Takara</td>
			<td>1068</td>
			<td></td>
		</tr>
		<tr>
			<td>LP</td>
			<td>Beyotime</td>
			<td>C0533</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium chloride</td>
			<td>APEXBio</td>
			<td>B6083</td>
			<td></td>
		</tr>
		<tr>
			<td>Malvern Zetasizer Nano ZS90</td>
			<td>Malvern</td>
			<td>NB007605</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidic chip</td>
			<td>ZHONGXINQIHENG</td>
			<td>Standard PDMS chip</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate readers</td>
			<td>ThermoFisher</td>
			<td>Varioskan lux</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>ThermoFisher</td>
			<td>ND-ONE-W (A30221)</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>ThermoFisher</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pseudouridine</td>
			<td>APE&#215;Bio</td>
			<td>B7972</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>Quanti-Luc</td>
			<td>InvivoGen</td>
			<td>Rep-qlc2</td>
			<td></td>
		</tr>
		<tr>
			<td>RiboRuler High Range RNA Ladder</td>
			<td>ThermoFisher</td>
			<td>SM1821</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free conical tube</td>
			<td>Biosharp</td>
			<td>BS-100-M</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>ThermoFisher</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Chemyx</td>
			<td>Fusion 101</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 transcription Kit</td>
			<td>Jinan</td>
			<td>E131</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient transfection of in vitro transcribed mrna in cultured cells using peptide-poloxamine nanoparticles

体外 转录的信使RNA（mRNA）疫苗在抗击2019年冠状病毒病（COVID-19）大流行方面显示出巨大的潜力。由于mRNA的易碎特性，mRNA疫苗中必须包含高效和安全的递送系统。自组装的肽-泊洛沙明纳米颗粒（PP-sNp）基因递送系统专为核酸的肺递送而设计，在介导成功的mRNA转染方面显示出有希望的能力。这里描述了一种制备PP-sNp的改进方法，以详细说明PP-sNp如何包封荧光素酶（MetLuc）mRNA并成功转染培养的细胞。甲基强质-mRNA是通过从线性DNA模板 的体外 转录过程获得的。通过使用微流体混合器将合成肽/泊洛沙明与mRNA溶液混合来产生PP-sNp，从而允许PP-sNp的自组装。随后通过测量zeta电位来评估PP-sNp的电荷。同时，采用动态光散射法测量了PP-sNp纳米粒子的多分散度和流体动力学尺寸.将mRNA / PP-sNp纳米颗粒转染到培养的细胞中，并测定细胞培养物中的上清液的荧光素酶活性。代表性结果证明了其 体外 转染能力。该协议可能有助于开发下一代mRNA疫苗递送系统。

重组质粒被消化以产生线性化的DNA模板（图2A）。使用所述方案，T7 体外 转录试剂盒每20μL反应可产生高达80-120μg的无上限甲基氯化钾-mRNA，每100μL反应可产生50-60μg封顶的甲基氯化物-mRNA。当用电泳分析时，具有高质量的完整MetLuc-mRNA应显示单个清晰的条带，如图 2B所示。从DNA模板引入反应中的污染物可能导致RNA降解和产量降低。
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Watch this Scientific Journal Video about Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles at JoVE.com

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

使用微流体混合装置开发自组装肽 - 泊洛沙明纳米颗粒（PP-sNp）以封装和递送 体外 转录的信使RNA。所描述的mRNA / PP-sNp可以在 体外有效地转染培养的细胞。

使用肽-泊洛沙明纳米颗粒在培养细胞中高效转染 体外 转录的mRNA

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. ...

肽-泊洛沙胺纳米颗粒是潜在的肺mRNA递送系统之一。我们的协议使肽-泊洛沙胺纳米颗粒具有更高的生产力和效率，这有助于粘膜mRNA疫苗的开发。该技术所需的材料成本低，协议可以在短时间内完成。肽-泊洛沙胺纳米颗粒具有介导高效mRNA转染的能力。肽-泊洛沙明纳米颗粒在人支气管上皮细胞中表现出成功的mRNA转染，这涉及粘膜COVID-19疫苗，肺癌治疗和囊性纤维化基因治疗的其他应用。我希望个人在开始进行实验之前，在相关领域以及协议和许多技术方面有一些基本知识。合成甲基荧光素酶的开放阅读框并将其克隆到pUC57载体中后，使用BamHI和KpnI在37摄氏度下制备线性化DNA模板1小时。如手稿中所述，使用 T7 转录试剂盒和假尿苷制备 20 微升体积的反应物用于 mRNA 合成。充分混合成分并在 37 摄氏度下孵育三小时。然后，通过在 65 摄氏度下加热 10 分钟来去除 50 微克未加帽 IVT mRNA 的二级结构。使用Cap 1加帽系统进行IVT mRNA加帽，并通过使用S-腺苷蛋氨酸和2'O-甲基转移酶在37摄氏度下修饰7-甲基鸟苷帽结构30至60分钟来制备Cap 1。用紫外可见分光光度计测量加帽IVT mRNA的浓度。使用 RNA 标记物，分析含有 18% 甲醛的 1% 甲醛变性琼脂糖凝胶中加帽 IVT mRNA 的分子量。通过将T704溶解在无核酸酶水中，制备每毫升10毫克的泊洛沙明704储备溶液。通过在无核酸酶水中溶解肽来制备每毫升2毫克肽储备溶液。将制备的溶液储存在4摄氏度。在冰上解冻IVT mRNA。在打开试管之前，将试管在室温下以300倍G离心三秒钟，并用无核酸酶的水稀释IVT mRNA溶液。通过将T704溶液稀释至每微升8微克浓度，将合成肽溶液稀释至0.555微克/微升浓度，分别用无核酸酶水制备T704和合成肽混合物溶液。在进一步使用之前，将混合溶液在室温下孵育15分钟。将IVT mRNA溶液吸入一毫升注射器中，避免注射器尖端的气隙或气泡。将注射器装入旋转块旁边的墨盒一侧。然后，用T704和合成肽混合物溶液填充一毫升注射器，并去除注射器尖端的任何气泡或气隙。将注射器放入泵的另一个入口，并将泵的总流量设置为每分钟4至10毫升。将十毫升无RNase的锥形管收集混合的IVT mRNA / PP-sNp溶液在混合装置的流路末端。运行泵开始混合，确保正确输入参数。运行泵六秒钟后，从锥形管中收集IVT mRNA / PP-sNp样品。转染前24小时将人支气管上皮细胞和树突状细胞系接种在96孔板中。在补充有10%热灭活胎牛血清和1%青霉素链霉素的培养基中培养细胞。除去生长培养基后，用每孔PBS0.2毫升洗涤铺板细胞。向含有铺板细胞培养物的每个孔中加入170微升无血清培养基。然后，向每个孔中加入30微升含有0.4微克甲基荧光素酶mRNA的IVT mRNA / PP-sNp制剂。用IVT mRNA / PP-sNp制剂在37摄氏度的潮湿5%富含二氧化碳的气氛中孵育细胞4小时。吸出转染培养基，加入0.2毫升新鲜培养基，补充有10%热灭活胎牛血清和1%青霉素链霉素。如前所述，将转染的细胞孵育24小时，并从每个孔中收集要检测的上清液。通过将PBS添加到腔肠素底物中来制备新鲜的测定溶液。涡旋10秒钟彻底混合溶液。将50微升收集的上清液加入96孔板中。设置酶标仪的读数时间为1， 000毫秒，并手动或通过自动进样向每个孔中加入30微升腔肠嗪溶液。将腔肠嗪溶液加入上清液后，立即单击开始以测量发光信号。分离并消化重组质粒以产生线性化的DNA模板。体外转录产生上加和封端的MetLuc mRNA。与转染市售脂质转染试剂、裸萤光素酶mRNA、PBS或T7044合成肽、人支气管上皮细胞中的混合溶液、16 HBE和树突状细胞系DC2.4萤光素酶mRNA/PP-sNp的细胞相比，转染的萤光素酶表达明显高于LP。 保护 MetLuc mRNA 免受降解，并促进外源性荧光素酶 mRNA 的转染效率。请确保成功去除未加帽的Met-mRNA的合成结构。仔细计算并下载 Met-mRNA T704 和肽化合物，以制备和压实泊洛沙明纳米颗粒。可以进行透射电子显微镜以观察肽-泊洛沙胺纳米颗粒的形态。该技术可帮助研究人员开发粘膜mRNA疫苗，并能够对每个相关细胞的基因治疗制剂进行高通量筛选。

Research

JoVE Journal

Immunology and Infection

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Seven Steps to Stellate Cells

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

高效转染树突状细胞的无细胞的成熟优化的协议

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

利用荧光蛋白可视化和定量<em&gt;衣原体</em&gt;液泡生长动态的活细胞

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

使用肽-泊洛沙明纳米颗粒在培养细胞中高效转染 体外 转录的mRNA

使用肽-泊洛沙明纳米颗粒在培养细胞中高效转染体外转录的mRNA