作者要感谢Gregg Sealy和Alain Conti的出色技术援助，以及柏林Max-Delbrück中心的Zsuzsanna Izsvák教授慷慨地提供了pSB100X和pT2-CAGGS-Venus质粒。这项工作得到了瑞士国家科学基金会和欧盟委员会在第七个框架方案的范围内的支持。Z.I由欧洲研究委员会ERC Advanced资助[ERC-2011-ADG 294742]。

<ol>
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作者没有什么可透露的。

这里提出的方案提供了一种分析转染细胞产生的PEDF和GM-CSF的抗氧化和保护功能的方法，可以应用于任何假定有益基因转染的细胞。在以通过移植转基因细胞将蛋白质递送到组织的基因治疗策略中，获得有关蛋白质表达水平，表达寿命以及表达蛋白质在疾病模型中的有效性的信息至关重要。在我们的实验室中，这里提出的方案可用于确定PEDF和GM-CSF对氧化应激的有效性，氧化应激已被假设为aAMD6，7发病机制中的重要元素。具体而言，我们使用该协议来定义SB100X介导的PEDF / GM-CSF转染的原代hRPE细胞的抗氧化作用。一些研究人员已经证明，H2O2诱导明显的氧化应激症状，但仍然允许细胞再生28，29，38，类似于我们的实验结果，该实验结果表明，350μM持续24小时在人ARPE-19和原代RPE细胞中诱导有效的氧化应激，可用于分析PEDF和GM-CSF的保护作用。H2O2作为氧化剂已被选择用于研究，因为它在眼睛中的生理存在和相应的防御机制，例如谷胱甘肽代谢20，21。我们的实验室已经检查了氧化应激的其他模型，例如用tBH处理细胞，其在氧化还原活性金属离子1的存在下启动脂质过氧化;然而，氧化应激可以忽略不计。在这里提出的实验中，细胞用H2O2处理24小时，因为我们发现2-6小时的较短处理时间足以诱导基因表达20的变化，但随后的后果，例如，细胞增殖，细胞活力和谷胱甘肽水平，可能还不可见。否则，细胞毒性和谷胱甘肽测定所需的小孔尺寸迅速导致汇合培养井;这可能导致接触抑制和氧化剂作用的掩盖。因此，与H2O2长期孵育似乎没有用，尽管在aAMD中看到的变性是由慢性氧化应激6，7引起的。
这里提出的实验的局限性是，接种的细胞数量会影响H 2O2的氧化作用，即对于相同的H2O2处理，当5，000个细胞接种时，观察到H2O2处理和未处理细胞之间谷胱甘肽水平的显着差异，而当10，000个细胞接种时则不然（图4).我们提出的方案需要接种少量细胞，即当细胞培养3天时为3，000个，当细胞培养2天时为5，000个（图1）。另一个限制是H2O2的浓度随着时间的推移而耗尽;Kaczara等人39已经表明，在ARPE-19细胞培养物中，H2O2在几个小时内会耗尽，这会影响慢性氧化应激模型的发展。这些研究人员提出了一种持续H2O2处理的替代方法，特别是使用葡萄糖氧化酶从培养基中的葡萄糖连续产生H 2 O2，但不能保证H 2 O2的标准化浓度。另一方面，我们建立的在单个脉冲中递送氧化剂的方案，与H2O2治疗必须重复数天的慢性模型相比，具有更快，更简单的优点38。
细胞抵消氧化损伤的能力取决于ROS产生和产生抗氧化剂的能力之间的平衡。在细胞中，三肽谷胱甘肽（GSH）是主要的还原剂，其可以被氧化成谷胱甘肽二硫化物（GSSG）并通过利用NADPH40的谷胱甘肽还原酶再生。在健康细胞中，超过90%的总谷胱甘肽池以还原形式存在。当细胞暴露于增加的氧化应激水平时，GSSG积累并且GSSG与GSH的比例增加。因此，监测生物样品中的谷胱甘肽氧化还原状态对于评估氧化应激和细胞损伤期间产生的自由基的细胞和组织的解毒状态至关重要。这里详述的谷胱甘肽定量方案足够灵敏，可以检测由RPE细胞转基因表达的PEDF和GM-CSF的抗氧化作用。
由于氧化应激影响线粒体活动40，特别有趣的是，PEDF对ROS水平的控制与线粒体解偶联蛋白2（UCP2）的调节有关，PEDF通过增加UCP2表达11，41来减弱氧化应激的影响。UCP2的主要功能是控制线粒体衍生的ROS并充当线粒体氧化应激的传感器41，42。在这里，除了检查PEDF和GM-CSF对谷胱甘肽水平的影响外，我们还具有UCP2的基因表达趋于增加（图7）;需要进一步的研究来确定PEDF和GM-CSF对UCP2基因表达的作用。
总体而言，目前的H2O2模型提供了一种全面的方法来研究基于转座子的基因疗法的有益效果，这些疗法旨在向患者的细胞提供抗氧化治疗基因，以将神经退行性疾病作为AMD进行治疗。

这里描述的模型为评估生物制药剂减少细胞氧化应激的效率提供了一种有用的方法。我们利用该模型研究了PEDF和GM-CSF对H2O2介导的氧化应激对视网膜色素上皮细胞的保护作用，这些细胞暴露于高水平的O2和可见光，以及光感受器外段膜的吞噬作用，产生显着水平的活性氧（ROS）1， 2.它们被认为是缺血性年龄相关性黄斑变性（aAMD）发病机制的主要贡献者3，4，5，6，7，8。此外，RPE合成的神经保护因子减少，特别是色素上皮衍生因子（PEDF），胰岛素样生长因子（IGF）和粒细胞巨噬细胞集落刺激因子（GM-CSF）导致RPE细胞的功能障碍和丢失，其次是光感受器和视网膜神经节细胞（RGC）死亡3，4，5.AMD是一种复杂的疾病，由代谢，功能，遗传和环境因素之间的相互作用引起4。缺乏对aAMD的治疗是工业化国家60岁以上患者失明的主要原因9，10。通过视网膜下移植过表达PEDF和GM-CSF的转基因RPE细胞重建神经保护性和神经源性视网膜环境，有可能通过减轻氧化应激的影响，抑制炎症和支持细胞存活来预防视网膜变性11，12，13，14，15，16.尽管有几种方法将基因递送到细胞，但我们选择了非病毒性过度活跃的睡美人转座子系统将PEDF和GM-CSF基因递送到RPE细胞，因为它的安全性，基因整合到宿主细胞的基因组中，以及它倾向于将递送的基因整合到非转录活性位点中，正如我们之前所展示的那样17， 18，19.
细胞氧化应激可以通过几种氧化剂在体外培养的细胞中诱导，包括过氧化氢（H2O 2），4-氢炔诺（HNE），过氧化氢叔丁酯（tBH），高氧张力和可见光（全光谱或紫外线照射）20，21。高氧张力和光需要特殊的设备和条件，这限制了向其他系统的可转移性。H 2 O2、HNE和 tBH 等试剂可诱导重叠的氧化应激分子和细胞变化。我们选择H2O2来测试PEDF和GM-CSF的抗氧化活性，因为它方便且具有生物学相关性，因为它是由RPE细胞在光感受器外段吞噬作用22期间作为活性氧中间体产生的，并且存在于体内的眼组织中23。由于谷胱甘肽的氧化可能是眼睛中H2O2产生部分原因，因此我们在研究中分析了GSH / 谷胱甘肽的水平，这与H2O2诱导的氧化应激和细胞的再生能力有关21，22。谷胱甘肽水平的分析特别相关，因为它参与眼睛的抗氧化保护机制24。暴露于H2O2经常被用作模型来检查RPE细胞1，25，26，27，28，29，30的氧化应激敏感性和抗氧化活性，此外，它显示出与光诱导的氧化应激损伤的相似性，氧化应激损伤是氧化应激21的"生理"来源。
为了评估神经保护因子的功能和有效性，我们开发了一种体外模型，该模型允许分析以量化由基因修饰以过度表达PEDF和GM-CSF的细胞表达的生长因子的抗氧化作用。在这里，我们表明，与PEDF和GM-CSF基因转染的RPE细胞比未转染的对照细胞更能抵抗H2O2的有害影响，如细胞形态学，密度，活力，谷胱甘肽的细胞内水平和UCP2基因的表达所证明的那样，UCP2基因编码线粒体解偶联蛋白2，已被证明可以减少活性氧（ROS）31。

<table><tbody><tr><td>24-well plates</td><td>Corning</td><td>353047</td><td></td></tr><tr><td>6-well plates</td><td>Greiner</td><td>7657160</td><td></td></tr><tr><td>96-well culture plate white with clear flat bottom&amp;nbsp;</td><td>Costar</td><td>3610</td><td>Allows to check the cells before measuring the luminescence (GSH-Glo Assay)</td></tr><tr><td>96-well plates</td><td>Corning&amp;nbsp;</td><td>353072</td><td></td></tr><tr><td>Acrylamid 40%</td><td>Biorad</td><td>161-0144</td><td></td></tr><tr><td>Amphotericin B</td><td>AMIMED</td><td>4-05F00-H</td><td></td></tr><tr><td>Antibody anti-GMCSF</td><td>ThermoFisher Scientific</td><td>PA5-24184</td><td></td></tr><tr><td>Antibody anti-mouse IgG/IgA/IgM&amp;nbsp;</td><td>Agilent</td><td>P0260</td><td></td></tr><tr><td>Antibody anti-PEDF&amp;nbsp;</td><td>Santa Cruz Biotechnology Inc</td><td>sc-390172</td><td></td></tr><tr><td>Antibody anti-penta-His&amp;nbsp;</td><td>Qiagen</td><td>34660</td><td></td></tr><tr><td>Antibody anti-phospho-Akt</td><td>Cell Signaling Technology</td><td>9271</td><td></td></tr><tr><td>Antibody anti-rabbit IgG H&amp;amp;L-HRP</td><td>Abcam</td><td>ab6721</td><td></td></tr><tr><td>Antibody donkey anti-rabbit Alexa Fluor 594&amp;nbsp;</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>A11034</td><td></td></tr><tr><td>Antibody goat anti-mouse Alexa 488</td><td>ThermoFisher Scientific</td><td>A-11029</td><td></td></tr><tr><td>ARPE-19 cell line</td><td>ATCC</td><td>CRL-2302</td><td></td></tr><tr><td>BSA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A9418-500G</td><td></td></tr><tr><td>chamber culture glass slides</td><td>Corning</td><td>354118</td><td></td></tr><tr><td>CytoTox-Glo Cytotoxicity Assay&amp;nbsp;</td><td>Promega</td><td>G9291</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542-5MG</td><td></td></tr><tr><td>DMEM/Ham`s F12&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8062</td><td></td></tr><tr><td>Duo Set ELISA kit</td><td>R&amp;amp;D Systems&amp;nbsp;</td><td>DY215-05</td><td></td></tr><tr><td>EDTA</td><td>ThermoFisher Scientific</td><td>78440</td><td></td></tr><tr><td>ELISAquant kit</td><td>BioProducts MD</td><td>PED613-10-Human</td><td></td></tr><tr><td>Eyes (human)</td><td>Lions Gift of Sight Eye Bank (Saint Paul, MN)</td><td></td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Brunschwig</td><td>P40-37500</td><td></td></tr><tr><td>Fluoromount Aqueous Mounting Medium</td><td>Sigma-Aldrich</td><td>F4680-25ML</td><td></td></tr><tr><td>FLUOstar Omega plate reader&amp;nbsp;</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>GraphPad Prism software (version 8.0)</td><td>GraphPad Software, Inc.</td><td></td><td></td></tr><tr><td>GSH-Glo Glutathione Assay</td><td>Promega</td><td>V6912</td><td></td></tr><tr><td>hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Merck</td><td>107209</td><td></td></tr><tr><td>ImageJ software (image processing program)</td><td>W.S. Rasband, NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/; 1997&amp;ndash;2014</td><td></td><td></td></tr><tr><td>Imidazol&amp;nbsp;</td><td>Axonlab</td><td>A1378.0010</td><td></td></tr><tr><td>Leica DMI4000B microscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>LightCycler 480 Instrument II&amp;nbsp;</td><td>Roche Molecular Systems</td><td></td><td></td></tr><tr><td>LightCycler 480 SW1.5.1 software</td><td>Roche Molecular Systems</td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>71376-1000</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Axonlab</td><td>3468.1000</td><td></td></tr><tr><td>Neon Transfection System&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>MPK5000</td><td></td></tr><tr><td>Neon Transfection System 10 &amp;micro;L Kit</td><td>ThermoFisher Scientific</td><td>MPK1096</td><td></td></tr><tr><td>Neubauer chamber</td><td>Marienfeld-superior</td><td>640010</td><td></td></tr><tr><td>Ni-NTA superflow&amp;nbsp;</td><td>Qiagen</td><td>30410</td><td></td></tr><tr><td>Nitrocellulose&amp;nbsp;</td><td>VWR</td><td>732-3197</td><td></td></tr><tr><td>Omega Lum G Gel Imaging System</td><td>Aplegen Life Science</td><td></td><td></td></tr><tr><td>PBS 1X</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Sigma-Aldrich</td><td>P0781-100</td><td></td></tr><tr><td>PerfeCTa SYBR Green FastMix</td><td>Quantabio</td><td>95072-012</td><td></td></tr><tr><td>PFA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>158127-100G</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>23227</td><td></td></tr><tr><td>Primers&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td></td><td>See Table 1 in Supplementary Materials</td></tr><tr><td>pSB100X (250 ng/&amp;micro;L)</td><td></td><td></td><td>M&amp;aacute;t&amp;eacute;s et al., 2009. Provide by Prof. Zsuzsanna Izsvak</td></tr><tr><td>pT2-CMV-GMCSF-His plasmid DNA (250 ng/&amp;micro;L)</td><td></td><td></td><td>Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.</td></tr><tr><td>pT2-CMV-PEDF-His plasmid DNA (250 ng/&amp;micro;L)</td><td></td><td></td><td>Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.</td></tr><tr><td>QIAamp DNA Mini Kit</td><td>QIAGEN</td><td>51304</td><td></td></tr><tr><td>recombinant hGM-CSF&amp;nbsp;</td><td>Peprotech</td><td>100-11</td><td></td></tr><tr><td>recombinant hPEDF&amp;nbsp;&amp;nbsp;</td><td>BioProductsMD</td><td>004-096</td><td></td></tr><tr><td>ReliaPrep RNA Cell Miniprep System</td><td>Promega</td><td>Z6011</td><td></td></tr><tr><td>RIPA buffer</td><td>ThermoFisher Scientific</td><td>89901</td><td></td></tr><tr><td>RNase-free DNase Set</td><td>QIAGEN</td><td>79254</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>QIAGEN</td><td>74204</td><td></td></tr><tr><td>SDS</td><td>Applichem</td><td>A2572</td><td></td></tr><tr><td>Semi-dry transfer system for WB&amp;nbsp;</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>SuperMix qScript</td><td>Quantabio</td><td>95048-025</td><td></td></tr><tr><td>Tris-buffered saline (TBS)&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>15504020</td><td></td></tr><tr><td>Triton X-100</td><td>AppliChem</td><td>A4975</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Sigma-Aldrich</td><td>T4174</td><td></td></tr><tr><td>Tween</td><td>AppliChem&amp;nbsp;</td><td>A1390</td><td></td></tr><tr><td>Urea</td><td>ThermoFisher Scientific</td><td>29700</td><td></td></tr><tr><td>WesternBright ECL HRP substrate</td><td>Advansta</td><td>K-12045-D50</td><td></td></tr><tr><td>Whatman nitrocellulose membrane</td><td>Chemie Brunschwig</td><td>MNSC04530301</td><td></td></tr></tbody></table>

induction and analysis of oxidative stress in sleeping beauty transposon-transfected human retinal pigment epithelial cells

氧化应激在几种退行性疾病中起着关键作用，包括年龄相关性黄斑变性（AMD），这是一种影响全球约3000万患者的病理。它导致视网膜色素上皮 （RPE） 合成的神经保护因子减少，例如色素上皮衍生因子 （PEDF） 和粒细胞-巨噬细胞集落刺激因子 （GM-CSF），随后 RPE 细胞丢失，最终导致光感受器和视网膜神经节细胞 （RGC） 死亡。我们假设通过转染的RPE细胞过表达PEDF和GM-CSF的视网膜下移植来重建神经保护性和神经源性视网膜环境，有可能通过减轻氧化应激的影响，抑制炎症和支持细胞存活来预防视网膜变性。使用 睡美人 转座子系统（SB100X），人RPE细胞已经转染了 PEDF 和 GM-CSF 基因，并使用qPCR，蛋白质印迹，ELISA和免疫荧光显示出稳定的基因整合，长期基因表达和蛋白质分泌。为了确认转染的RPE细胞分泌的 PEDF 和 GM-CSF 的功能和效力，我们开发了一种体外测定法来量化培养物中H2O2诱导的氧化应激对RPE细胞的减少。通过分析细胞形态、密度、谷胱甘肽的细胞内水平 、UCP2 基因表达和细胞活力来评估细胞保护。与未处理的对照相比，转染的RPE细胞过度表达PEDF和/或GM-CSF以及未转染但用PEDF和/或GM-CSF（市售或从转染细胞中纯化）预处理的细胞显示出显着的抗氧化细胞保护。目前的H2O2模型是一种简单有效的方法来评估可能有效治疗AMD或类似神经退行性疾病的因子的抗氧化作用。

诱导人视网膜色素上皮细胞中的氧化应激 ARPE-19和原代hRPE细胞用不同浓度的H2O2处理24小时，并定量抗氧化剂谷胱甘肽的细胞内水平（图2A，B）。H2O2在50μM和100μM不影响谷胱甘肽的产生，而在350μM时，ARPE-19和原代hRPE细胞中的谷胱甘肽显着降低。细胞毒性分析表明，350μM是H2O2的最低浓度，导致细胞活力显着降低（图2C）。从形态学上讲，用H 2 O2处理的ARPE-19细胞看起来扩散更少，更圆润，随着H 2 O2浓度的增加，特征变得更加明显（图3）。对于用H2O2处理的PEDF-和GM-CSF转染细胞，效果不太突出（图3）。为了证明细胞数对H2O2介导的氧化应激的影响，将每孔5，000和10，000个ARPE-19细胞接种在白色96孔板中;第二天，用350μM H2O2处理细胞24小时，测定谷胱甘肽的水平。图4显示谷胱甘肽的水平仅在接种了5，000个细胞的孔（n = 3）中降低。对于确定H 2O2产生的ROS的抗氧化剂作用的实验，必须考虑细胞的数量;对于本报告中提出的特定方案，用350μM H 2 O 2处理24小时的3，000-5，000个细胞/孔（96孔板）适合显示显着的细胞损伤，同时保留恢复能力，模仿对氧化应激诱导的细胞损伤的亚急性反应。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61957/61957fig02.jpg"> 图2:在用H 2 O 2处理的人RPE细胞中，作为谷胱甘肽水平和细胞活力的氧化应激水平证明.（A）ARPE-19细胞暴露于几种浓度的H 2 O 2的细胞中，与H 2O2相比，在350μM（0.66μM），500μM（0.022μM）和700μM（0.002μM）的谷胱甘肽水平（括号中）显示出显着降低谷胱甘肽水平（括号中）（p<0.0001为350， 500 和 700 μM H2O2）。（B） 原代人RPE细胞显示谷胱甘肽水平下降;然而，与ARPE-19相比，效果不那么突出，但与350，500和700μM H2O2的对照组相比，仍然具有统计学意义。350μM是最低的H2O2浓度，产生显着的氧化损伤，如谷胱甘肽水平与未处理的对照细胞相比降低（p = 0.0022）。谷胱甘肽水平随着H2O2浓度的增加而降低（500μM:p = 0.022;700μM:p = 0.0005）。（C）细胞毒性分析表明，350μM H2O2是产生活细胞百分比显着降低的最低浓度（350，500和700μM的p<0.0001）。数据以SD±平均值表示（n = 3次重复），显著差异表示为（*）;使用Tukey的多重比较测试对C-与H 2 O2-处理组进行比较，对方差分析进行事后计算。C-:H2O2未处理的细胞。该图已从Bascuas等人37修改而来。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61957/61957fig02large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61957/61957fig03.jpg"> 图3:用H 2 O2处理的非转染和PEDF或GM-CSF转染ARPE-19细胞的形态学。用增加浓度的H2O2处理的细胞在培养孔中显示出较少的细胞，并显示出更圆润，更少的扩散形态，这是细胞应激的已知迹象。请注意，对于PEDF或GM-CSF转染的细胞，细胞应激不那么突出，并且生长类似于未处理的对照细胞。C-:未经处理的对照细胞。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61957/61957fig03large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61957/61957fig04.jpg"> 图4:细胞数量对H2O2诱导的氧化应激的影响。将5，000和10，000个ARPE-19细胞/孔接种在96孔板中。24小时后，用350μM H2O2处理细胞24小时。在接种了5，000个细胞（p = 0.031，t-test）的孔中观察到谷胱甘肽水平的显着差异，但在接种了10，000个细胞的孔中却没有观察到谷胱甘肽水平的显着差异。C-:未经处理的细胞。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61957/61957fig04large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
SB100X转染人RPE细胞在氧化应激条件下对PEDF和GM-CSF的抗氧化作用分析 作为阳性对照，ARPE-19和原代人RPE细胞在24小时H 2 O 2处理之前和期间用5，50或500ng / mL市售PEDF或GM-CSF处理2天。与在氧化条件下（H 2O2处理）下未经处理的对照相比，用500ng / mL PEDF或50 ng / mL GM-CSF处理的ARPE-19细胞产生显着更多的谷胱甘肽（图5A）;从转染ARPE-19细胞的培养基中纯化的PEDF和GM-CSF显示出类似的效果（图5B）。在原代hRPE细胞中，加入500 ng / mL PEDF，50 ng / mL GM-CSF或500 ng / mL PEDF加50 ng / mL GM-CSF，无论是商业的还是从PEDF或GM-CSF转染的ARPE-19细胞调节的培养基中纯化的，都减少了细胞损伤，这反映在谷胱甘肽水平显着增加（图5C）。与对照细胞相比，用转染ARPE-19细胞的调节培养基处理10天的原代hRPE细胞也显示出更高的谷胱甘肽水平（图5D）。基于这些结果，已经进行了进一步的实验，PEDF为500 ng / mL，GM-CSF为50 ng / mL。
ARPE-19和原代hRPE细胞使用 睡美人 转座子系统结合电穿孔转染PEDF和/或GM-CSF的基因。通过RT-qPCR，WB，ELISA和免疫组织化学（参见 补充材料， 图S1和 图S2）转染并分析基因表达后，转染的ARPE-19细胞暴露于350μM H2O2 24 小时后，谷胱甘肽水平明显高于未转染的H2O2处理细胞（图6A).对于原代hRPE细胞，当所有供体都包括在分析中时，与用H2O2 处理的非转染细胞相比，PEDF转染细胞中的谷胱甘肽水平显着增加。此外，供体2和3显示所有转染组（PEDF，GM-CSF，PEDF和GM-CSF）的谷胱甘肽水平显着增加（数据未显示）。
UCP2基因表达的研究通过检查线粒体氧化应激完成了分析。在转染的ARPE-19细胞中进行了概念验证系列，用350μM H2O2处理24小时。如图7所示，在转染的ARPE-19细胞中，H 2 O2处理后UCP2基因表达水平升高，但增加不显著。图8显示了暴露于H2O2的GM-CSF转染细胞裂解物的磷酸化Akt（pAkt）的WB;归一化数据显示，与未处理的对照相比，仅略有下降，表明GM-CSF可以保护细胞免受氧化应激损伤。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61957/61957fig05.jpg"> 图5:谷胱甘肽水平作为PEDF和GM-CSF抗氧化能力的标志物（A）在24小时H2O 2暴露之前和期间用500 ng / mL PEDF或50 ng / mL GM-CSF处理ARPE-19细胞3天，将谷胱甘肽水平从0.83μM（C）增加到1.83μM（PEDF）和1.3μM（GM-CSF）， p = 0.026 和 p = 0.031。在5 ng / mL的浓度下，没有观察到谷胱甘肽的增加;对于PEDF或GM-CSF，谷胱甘肽水平在50至500ng / mL之间的差异均不显着。（B） 从转染 ARPE-19 细胞的条件培养基中纯化的 PEDF （500 ng/mL） 和 GM-CSF （50 ng/mL） 显示出与市售 PEDF 或 GM-CSF 相似的效果（p = 0.018，方差分析）。（C） 在 24 小时 H 2 O 2 处理之前和期间，向原代 hRPE 细胞培养基中加入 500 ng/mL PEDF、50 ng/mL GM-CSF 或 500 ng/mL PEDF 加50 ng/mL GM-CSF，显著提高了用 PEDF 处理的细胞中谷胱甘肽的水平（2.6 μM [商业]， 2.5μM [纯化]），GM-CSF（2.9μM [商业]，3.3μM [纯化]）和PEDF加GM-CSF（3.0μM [商业]，2.9μM [纯化]）与未处理的细胞（1.9μM）相比（p = 0.006，Kruskal-Wallis测试）。（D）在用H 2 O2处理细胞之前，在PEDF，GM-CSF或PEDF-GM-CSF转染的ARPE-19细胞的调节培养基中培养10天的hRPE细胞观察到谷胱甘肽水平显着增加（p =0.003，Kruskal-Wallis测试）（数据显示为一个供体）。数据表示为 SD ±平均值（n = 3 次重复）。显著差异用 （*） 表示;通过计算Tukey或Dunnett的多重比较测试来执行方差分析的事后计算，将"C"与PEDF-/GM-CSF处理组进行比较。C:仅用H2O2处理的细胞，P:用PEDF处理的细胞，G:用GM-CSF处理的细胞，P + G:用PEDF加GM-CSF处理的细胞。该图已从Bascuas等人37修改而来。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61957/61957fig05large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61957/61957fig06.jpg"> 图6:谷胱甘肽水平作为PEDF和GM-CSF转染的人RPE细胞的抗氧化能力的标志物显着高于非转染细胞（1.9μM），即PEDF和GM-CSF转染细胞的3.0μM，转染的ARPE-19细胞的谷胱甘肽水平暴露于350μM H2O 24小时（转染后56天）， 和3.4μM用于双转染细胞（p = 0.0001，方差分析）。数据表示为 SD ±平均值（n = 3 次重复）。（B） 点阵图显示了四种不同供体的平均谷胱甘肽值（C: 0.77 μM;P: 1.45 μM;G: 1.16 μM;P + G:1.2μM），这在未转染和PEDF转染的细胞之间显着差异（p = 0.028，使用Tukey的多重比较测试进行方差分析的事后计算，比较"C"与PEDF-/GM-CSF处理组）。当单独分析供体时，供体N°2和N°3（参见图中符号的表2）与用H2O2处理的非转染对照组（未显示显著性）相比，所有转染组均显示出显着差异。C:非转染细胞，P:PEDF转染细胞，G:GM-CSF转染细胞，P+G:PEDF和GM-CSF转染细胞。该图已从Bascuas等人37修改而来。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61957/61957fig06large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61957/61957fig07.jpg"> 图7:用H 2 O 2处理的转染ARPE-19细胞中的UCP2基因表达。由于UCP2基因表达可用于检查线粒体氧化损伤，因此我们检查了转染ARPE-19细胞对PEDF和GM-CSF的过表达的影响。与未转染的对照组相比，用H2O 2处理的转染ARPE-19细胞虽然没有统计学意义，但与未转染的对照相比，UCP2基因表达增加，表明氧化应激降低，PEDF-为1.57，GM-CSF-为1.51，PEDF-加GM-CSF转染细胞为2.36。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61957/61957fig07large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61957/61957fig08.jpg"> 图8:来自GM-CSF转染ARPE-19细胞裂解物的磷酸化Akt（Ser473）的蛋白质印迹。WB证明，GM-CSF在未经处理和H2O2处理的培养物中增强Akt的磷酸化（UT:3.32;H2O2: 2.69）.这些值归一化为未转染的非H2O2处理的细胞（C / UT）。C:未转染，G:GM-CSF转染细胞，UT:未用H2O2处理的细胞，H2O2:用H2O2处理的细胞。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61957/61957fig08large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
表S1:用于RT-qPCR的引物对序列和退火时间/温度。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61957/Table%20S1_20201019.xlsx" style="font-size: 14px;" target="_blank">请点击此处下载此表格。</a>
图S1:转染hRPE细胞中的 PEDF 和 GM-CSF 基因表达分析。 RT-qPCR证实，与未转染的细胞相比，转染原代hRPE细胞的 PEDF（p = 0.003，Kruskal-Wallis试验）和 GM-CSF（p = 0.013，Kruskal-Wallis试验）基因表达显着增加。2^（-ΔΔCT）方法在本例中使用36。数据表示为SD±平均值（n = 4个供体）。每个点表示三个重复的平均值。该图已从Bascuas等人37修改而来。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61957/Figure%20S1.tif" style="font-size: 14px;" target="_blank">请点击此处下载此文件。</a>
图S2:转染原代hRPE和ARPE-19细胞中的蛋白质分泌。 （A）ELISA对分泌蛋白的定量表明，转染的hRPE细胞比未转染的细胞分泌更多的PEDF和GM-CSF（PEDF为p = 0.014，GM-CSF为p = 0.006，Kruskal-Wallis测试）。数据以SD±平均值表示（n = 4个供体）。每个点表示三个重复的平均值。（B） PEDF-GM-CSF双重染色证实了PEDF和GM-CSF在双转染ARPE-19细胞中的共分泌（合并图）。该图已从Bascuas等人37修改而来。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61957/Figure%20S2.tif" style="font-size: 14px;" target="_blank">请点击此处下载此文件。</a>
补充材料。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61957/Supplementary%20material_JoVE_20201124.docx" style="font-size: 14px;" target="_blank">请点击此处下载此文件。</a>

Watch this Scientific Journal Video about Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells at JoVE.com

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

我们提出了一种开发和使用氧化应激模型的方案，方法是用H2O2处理视网膜色素上皮细胞，分析细胞形态，活力，密度，谷胱甘肽和UCP-2水平。它是研究转座子转染细胞分泌的蛋白质治疗神经视网膜变性的抗氧化作用的有用模型。

睡眠美人转座转染人视网膜色素上皮细胞氧化应激的诱导与分析

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

induction-analysis-oxidative-stress-sleeping-beauty-transposon

Nanyang Technological University

Research

JoVE Journal

Medicine

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

2.5K 次观看.  University of Geneva. 我们提出了一种开发和使用氧化应激模型的方案，方法是用H2O2处理视网膜色素上皮细胞，分析细胞形态，活力，密度，谷胱甘肽和UCP-2水平。它是研究转座子转染细胞分泌的蛋白质治疗神经视网膜变性的抗氧化作用的有用模型。

视频: Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

用基因编码荧光传感器评估毒理学氧化应激的成像方法

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

科研

癌症研究

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

使用实时成像可视化氧化损伤对果蝇卵室的影响

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

发育生物学

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

在 vivo小鼠创面模型中活性氧的成像

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

免疫与感染

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

生物学

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

从鸡胚胎的直膜到研究棒到锥细胞相互作用的锥形丰富文化

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

神经科学

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during development and in disease states. Single-cell RNA sequencing allows us to comprehensively profile gene expression changes observed in retinal development and disease at a cellular level, while single-cell ATAC-Seq allows analysis of chromatin accessibility and transcription factor binding to be profiled at similar resolution. Here the use of these techniques in the developing retina is described, and MULTI-Seq is demonstrated, where individual samples are labeled with a modified oligonucleotide-lipid complex, enabling researchers to both increase the scope of individual experiments and substantially reduce costs.

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

使用 scRNA-Seq 和 scATAC-Seq 对视网膜基因表达和染色质可及性进行多重分析

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, &#946;-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since M&#252;ller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe (DCFH-DA) in M&#252;ller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible.

使用2'，7'-二氯荧光素双乙酸探针和流式细胞术在穆勒神经胶质细胞中定量活性氧

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

使用睡美人转座子系统对原代人色素上皮细胞进行基于电穿孔的遗传修饰

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

医学

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.