（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测
Summary
监控活细胞的泛素 – 蛋白酶体活性的方法。一个degron不稳定的绿色荧光蛋白（GFP-DGN）和一个稳定的的GFP-dgnFS融合蛋白的生成和使用慢病毒表达载体导入细胞。这种技术允许产生稳定的GFP-dgn/GFP-dgnFS表达细胞株中，泛素 – 蛋白酶体活性可以很容易地使用的反射荧光或流式细胞术评估。
Abstract
蛋白酶是异常的，错误折叠的，损坏或氧化蛋白1，2中的蛋白水解降解的主要涉及的胞内细胞器。蛋白酶体活性的维护被牵连了许多关键的细胞过程，如细 胞的应激反应，细胞周期调控和细胞分化或免疫系统的反应5。已与泛素-蛋白酶体系统的功能障碍，肿瘤，神经退行性疾病4，6的发展。此外，在蛋白酶体活性的减少被发现作为一个功能的细胞衰老和有机体的老化7，8，9，10。在这里，我们提出了一种方法来衡量使用GFP-的DGN融合蛋白在活细胞的泛素 – 蛋白酶体的活性。至能够监视泛素 – 蛋白酶体活性的活的原代细胞，互补的DNA构建体编码的绿色荧光蛋白（GFP）-DGN融合蛋白（GFP-DGN，不稳定）和携带的移码突变的变体（GFP-dgnFS的，稳定11）被插入在慢病毒表达载体。我们更喜欢这种技术比传统的转染技术，因为它保证了一个非常高的转染效率的细胞类型或捐赠者的年龄无关。可以使用显示在GFP-dgnFS（稳定）蛋白和蛋白酶体抑制剂的存在或不存在中的不稳定的蛋白（GFP-DGN）的荧光之间的差异，来估计在每个特定的细胞株的泛素 – 蛋白酶体活性。这些差异可以通过荧光显微镜监测，也可以通过流式细胞仪检测。
Protocol
1。质粒的构建订购定制寡核苷酸编码为DGN（ACKNWFSSLSHFVIHL 11）和为dgnFS（HARTGSLACPTSSSICE）和结扎到质粒pEGFP-C1载体，以获得与DGN / dgnFS的（ 图1）的GFP的融合。 通过PCR扩增的GFP-dgn和GFP-dgnFS的的编码序列，。根据协议pENTR定向TOPO克隆试剂盒，并继续与pLenti6/V5定向TOPO克隆试剂盒（ 图6）。 2。病毒生产转染前一天（第1天?…
Discussion
用绿色荧光蛋白（GFP）作为底物泛素-蛋白酶体活性记者，发表于2000年12首次出版。从那时起，GFP已成为一种普遍的工具，可视化细胞的活动，特别是泛素 – 蛋白酶体的过程。若要监视在体内的泛素-蛋白酶体活性的转基因小鼠模型与基于GFP的记者已被引入13。额外另一种转基因小鼠模型的体内研究建立一个类似的不稳定degron的GFP记者作为本出版物中的14。
<p…Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究由国家老化（NFN S93）由奥地利科学基金会（FWF），欧盟委员会综合项目MiMAGE和PROTEOMAGE，荷兰基因组计划研究网络/荷兰科学研究组织（NGI / NWO; 05040202，050 – 060-810恩查），欧盟资助网络的卓越寿命（FP6 036894），和基因组学创新研究计划（SenterNovem IGE01014和IGE5007）。
Materials
| Name of the reagent | Company | Catalogue number |
| pEGFP-C1 Vector | BD Bioscience Clontech | 6084-1 |
| pENTR Directional TOPO Cloning Kit | Invitrogen | K2400-20 |
| pLenti6/V5 Directional TOPO Cloning Kit | Invitrogen | V496-10 |
| Lipofectamine 2000 Reagent | Invitrogen | 11668019 |
| DMEM | Sigma | D5546 |
| PVDF filter (Rotilabo-Spritzenfilter) | Roth | P667.1 |
| Polyethylene glycol | Sigma | P2139 |
| NaCl | Merck | 1.06404.1000 |
| Dulbecco’s Phosphate Buffered Saline 1x (PBS) | Invitrogen | 14190 |
| hexadimethrine bromide | Sigma | 10,768-9 |
| Blasticidin | Invitrogen | R21001 |
| Crystal violet | Sigma | C3886 |
| FACS tubes | BD Biosciences | |
| Penicillin Streptomycin (Pen-Strep) | Invitrogen | 15140130 |
| L-glutamine 200 mM | Invitrogen | 25030024 |
| Fetal Bovine Serum (FBS) | Biochrom AG | S0115 |
| MEM Non-Essential Amino Acids (NEAA) 100x | Invitrogen | 11140035 |
| MEM Sodium Pyruvate 100 mM | Invitrogen | 11360039 |
| D-(+)-Glucose (45%) | Sigma | G8769 |
| Geneticin | Invitrogen | 11811023 |
| CaCl2 | Merck | C5080 |
| Hepes | Sigma | H3375 |
| Trypsin-EDTA (0.05%) | Invitrogen | 25300054 |
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Tags
Ubiquitin-proteasome ActivityDegronGreen Fluorescent Protein (GFP)Reporter ProteinProteolysisIntracellular OrganelleAbnormal ProteinsMisfolded ProteinsDamaged ProteinsOxidized ProteinsProteasome MaintenanceCellular ProcessesStress ResponseCell Cycle RegulationCellular DifferentiationImmune System ResponseUbiquitin-proteasome System DysfunctionTumorsNeurodegenerative DiseasesCellular SenescenceOrganismal AgingPrimary CellsLentiviral Expression VectorsTransfection Efficiency
Citer Cet Article
Greussing, R., Unterluggauer, H., Koziel, R., Maier, A. B., Jansen-Dürr, P. Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein. J. Vis. Exp. (69), e3327, doi:10.3791/3327 (2012).