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Cellular Translational Reporter Assay
细胞翻译报告基因分析   

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Abstract

The method described here allows measuring the effect of exogenously introduced modifications to in vitro-transcribed mRNA on the translation in cells. Using cells derived from knockout mice and control littermates, this method enables to compare the results in the presence or absence of specific gene products. In our lab, we used this protocol to check whether the exogenous addition of 5’ capping and 2’-O methylation to in vitro-mRNA affects the translational efficiency. Here we describe the details of our experiments.

Materials and Reagents

  1. Mouse embryonic fibroblasts (prepared from day 14.5 embryos)
  2. Vector pGL4.14 (Luc2 encoding vector) (Promega Corporation, catalog number: E6691 )
  3. Primers (Invitrogen custom DNA primers)
    1.  5’-TAATACGACTCACTATAGGCCACCATGGAAGATGCCAAAAA-3’ (The T7 class III promoter sequence is underlined.)
    2.  5’-TACCACATTTGTAGAGGTTTTACTTGCTTT-3’
  4. rTaq DNA polymerase (TOYOBO, catalog number: TAP-211 )
  5. Agarose gel
  6. Ethidium bromide
  7. Illustra GFX PCR and Gel Band Purification Kit (GE, catalog number: 28-9034-71 )
  8. MEGAScript In vitro transcription Kit (Life Technologies, Ambion®, catalog number: AM1333 )
    Note: Nuclease-free Water and LiCl Precipitation Solution are included in the kit.
  9. 80% ethanol
  10. ScriptCap m7G capping system (EpiCentre, catalog number: SCCE0610 )
  11. ScriptCap 2’-O-Methyltransferase Kit (EpiCentre, catalog number: SCMT0610 )
  12. RNeasy Mini Kit (QIAGEN, catalog number: 74104 )
  13. Opti-MEM I reduced serum medium (Life Technologies, Gibco®, catalog number: 31985-070 )
  14. D-PBS(-) (Nacalai Tesque, catalog number: 14249-95 )
  15. Lipofectamine 2000 DNA Transfection Reagent (Life Technologies, catalog number: 11668-019 )
  16. Dual-luciferase reporter assay system (Promega Corporation, catalog number: E1960 )
  17. BCA protein assay reagent (Thermo Fisher Scientific, catalog number: 23227 )

Equipment

  1. Cell scraper
  2. GeneAmp PCR system 9700 (Applied Biosystems®)
  3. Centrifugal Concentrator CC105 (TOMY)
  4. Lumat LB 9507 Luminometer (Berthold Technologies)
  5. Model 680 Microplate Reader (Bio-Rad Laboratories)

Procedure

  1. Subcloning of Luc2 cDNA
    1. Prepare the polymerase chain reaction mix.
      Mix following components on ice:
      0.2 μl of rTaq DNA polymerase
      5 μl of 10x Buffer (+Mg) for rTaq
      5 μl of dNTPs (2 mM each)
      4 μl of primers (10 mM each of Forward and Reverse primers)
      1 μl of vector pGL4.14 (100 ng)
      35 μl of water
    2. Run the polymerase chain reaction.
      Place the reaction mix to the GeneAmp PCR system 9700.
      PCR program:
      Step 1: 94 °C 5 min
      Step 2: 94 °C 30 sec
      Step 3: 60 °C 2 min
      Step 4: 74 °C 1 min (repeat steps 2-4 for 35 times)
      Step 5: 74 °C 10 min
    3. Purification of PCR amplified fragments.
      1. Electrophorese the PCR products on 1% of agarose gel.
      2. Stain Gel with Ethidium Bromide.
      3. Cut the gel region including an amplified DNA fragment (approximately 1.6-kbp).
      4. Purify PCR amplified DNA from gels using illustra GFX PCR and Gel Band Purification Kit.
        Note: In this process, we elute DNA fragment with 30 μl of Nuclease-free Water (8 μl aliquots of the eluent are used as a template DNA for following in vitro transcription).

  2. In vitro transcription of Luciferase mRNA under the control of T7 promoter
    1. Prepare the in vitro transcription mix.
      Mix following components at room temperature:
      8 μl of Template DNA
      2 μl of 10x Reaction Buffer
      2 μl of 10 mM ATP
      2 μl of 10 mM CTP
      2 μl of 10 mM UTP
      2 μl of 10 mM GTP
      2 μl of T7 Enzyme Mix
      Mix thoroughly with pipetting.
    2. Incubate at 37 °C, 4 h.
    3. Add 2 μl of TURBO DNase (a component of MEGAScript) to digest the rest template DNA and incubate 15 min at 37 °C on the block incubator.
    4. RNA extraction with lithium chloride (LiCl) precipitation.
      1. Add 30 μl of nuclease-free water and 30 μl of LiCl precipitation solution to the in vitro transcription products.
      2. Mix thoroughly with vortex mixer. Chill for 1 h at -20 °C.
      3. Centrifuge at 4 °C for 15 min at 15,000 rpm.
      4. Discard the supernatants, and wash the pellet with 1 ml of 80% ethanol.
      5. Centrifuge at 4 °C for 15 min at 15,000 rpm.
      6. Discard the supernatants, and dry the pellet with centrifugal concentrator CC105 for 5 min.
      7.  Resuspend the RNA in 20 μl of Nuclease-free Water, and stored at -80 °C.

  3. Enzymatic modification of RNA 5’ end
    1. Add m7G cap, or m7G cap and 2’-O methylation to the in vitro-transcribed RNA with ScriptCap system.
      1. Adjust the volume of transcribed RNA (50 μg) to 67 μl with Nuclease-free Water.
      2. Denature the RNA at 65 °C for 10 min, then transfer the tube immediately to ice.
      3. While the RNA is denaturing, mix following components.
        10 μl of 10x ScriptCap Capping Buffer
        10 μl of 10 mM GTP
        2.5 μl of 20 mM SAM
        2.5 μl of ScriptGuard RNase Inhibitor
        4 μl of ScriptCap 2’-O-Methyltrasferase (100 U/μl) for 2’-O methylated 5’ capped RNA or Nuclease-free Water for 2’-O unmethylated 5’ capped RNA
      4. Add 4 μl of ScriptCap Capping Enzyme (10 U/μl) and 67 μl of denatured RNA to the reaction cocktail from step C1c.
      5. Incubate at 37 °C for 2 h.
      6. Reaction products were purified with an RNeasy Mini Kit.

  4. RNA transfection with mouse embryonic fibroblasts
    1. Seed a 35 mm diameter dish with 2 x 105 mouse embryonic fibroblasts in 1 ml DMEM, and incubate 24 h.
    2. Transfect RNA using Lipofectamine 2000.
      1. Dilute 10 μl of Lipofectamine 2000 DNA Transfection Reagent in 240 μl of Opti-MEM medium per sample.
      2. Dilute 2 μg of luciferase RNA in 250 μl of Opti-MEM medium.
      3. Add 250 μl of diluted Lipofectamine 2000 DNA Transfection Reagent to 250 μl of diluted Luciferase RNA. Mix thoroughly.
      4. Incubate at room temperature for 15 min.
      5. Add RNA-lipid complex to the mouse embryonic fibroblasts, and incubate for 6 h.

  5. Luciferase translational reporter assays
    1. Preparation of cell lysates.
      1. After incubation for 6 h, wash the RNA-transfected mouse embryonic fibroblasts with D-PBS(-) twice.
      2. Add 100 μl of 1x passive lysis buffer and harvest cells with cell scraper.
      3. Centrifuge at 4 °C for 15 min at 15,000 rpm.
      4. Determine the protein concentration in 5 μl aliquots of the supernatants by BCA Protein Assay.
      5. Add equal protein amount (determined by BCA protein assay) of aliquots of the supernatants to 50 μl of Luciferase Assay Reagent II (LAR II).
      6.  Immediately, measure the relative luciferase units (RLUs) on Lumat LB 9507 Luminometer according to the manufacturer’s instruction.


        Figure 1. Luciferase activity of introduced RNAs. Wild-type and Ifit1-deficient (Ifit1-/-) mouse embryonic fibroblasts were transiently transfected with three different types of luciferase mRNAs (5’-uncapped, 5’ capped but 2’-O unmethylated; 5’ cap+/2’-O Me-, 5’ capped and 2’-O methylated; 5’ cap+/2’-O Me+). Luciferase activities are shown as relative light units (RLU), and the numbers of RLU were normalized by the concentrations of proteins determined in step E1d. Data are shown as means ± SDs of triplicate samples. These data show that Ifit1 selectively inhibits the translation of 5’ capped but 2’-O unmethylated (5’ cap+/2’-O Me-) luciferase mRNA.

Acknowledgments

This work was adapted from the following paper: Kimura et al. (2013). This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, the Japan Science and Technology Agency, and by the Ministry of Health, Labour and Welfare.

References

  1. Kimura, T., Katoh, H., Kayama, H., Saiga, H., Okuyama, M., Okamoto, T., Umemoto, E., Matsuura, Y., Yamamoto, M. and Takeda, K. (2013). Ifit1 inhibits Japanese encephalitis virus replication through binding to 5′ capped 2′-O unmethylated RNA. J Virol 87(18): 9997-10003.

简介

本文所述的方法允许测量外部引入的修饰对体外转录的mRNA对细胞翻译的影响。 使用来自敲除小鼠和对照同窝出生的细胞,该方法能够比较存在或不存在特定基因产物的结果。 在我们的实验室中,我们使用该协议来检查外部添加5'帽化和2'-O甲基化在体外 -mRNA是否影响翻译效率。 这里我们描述我们的实验的细节。

材料和试剂

  1. 小鼠胚胎成纤维细胞(从第14.5天胚胎制备)
  2. 载体pGL4.14( Luc2 编码载体)(Promega Corporation,目录号:E6691)
  3. 引物(Invitrogen定制DNA引物)
    1. 5'-TAATACGACTCACTATAG GCCACCATGGAAGATGCCAAAAA-3'(T7 III类启动子序列加下划线。)
    2.   5'-TACCACATTTGTAGAGGTTTTACTTGCTTT-3'
  4. rTaq DNA聚合酶(TOYOBO,目录号:TAP-211)
  5. 琼脂糖凝胶
  6. 溴化乙锭
  7. Illustra GFX PCR和凝胶带纯化试剂盒(GE,目录号:28-9034-71)
  8. MEGAScript体外转录试剂盒(Life Technologies,Ambion ,目录号:AM1333)
    注意:试剂盒中包含无核酸酶的水和LiCl沉淀溶液。
  9. 80%乙醇
  10. ScriptCap m7G加盖系统(epicenter,目录号:SCCE0610)
  11. ScriptCap 2'-O-甲基转移酶试剂盒(震中,目录号:SCMT0610)
  12. RNeasy迷你包(QIAGEN,目录号:74104)
  13. Opti-MEM I还原血清培养基(Life Technologies,Gibco ,目录号:31985-070)
  14. D-PBS( - )(Nacalai Tesque,目录号:14249-95)
  15. Lipofectamine 2000DNA转染试剂(Life Technologies,目录号:11668-019)
  16. 双荧光素酶报告基因测定系统(Promega Corporation,目录号:E1960)
  17. BCA蛋白测定试剂(Thermo Fisher Scientific,目录号:23227)

设备

  1. 细胞刮刀
  2. GeneAmp PCR系统9700(Applied Biosystems )
  3. 离心式浓缩器CC105(TOMY)
  4. Lumat LB 9507发光计(Berthold Technologies)
  5. Model 680微孔板读数器(Bio-Rad Laboratories)

程序

  1. 亚克隆Luc2 cDNA
    1. 准备聚合酶链反应混合物。
      在冰上混合以下组分:
      0.2μlrTaq DNA聚合酶
      5μl用于rTaq的10x缓冲液(+ Mg)
      5μldNTP(每种2mM)
      4μl引物(每种正向和反向引物各10mM)
      1μl载体pGL4.14(100ng)
      35μl水
    2. 运行聚合酶链反应。
      将反应混合物置于GeneAmp PCR系统9700.
      PCR程序:
      步骤1:94℃5分钟
      步骤2:94℃30秒
      步骤3:60℃2分钟
      步骤4:74°C 1分钟(重复步骤2-4,35次)
      步骤5:74℃10分钟
    3. PCR扩增片段的纯化
      1. 在1%的琼脂糖凝胶上电泳PCR产物
      2. 用溴化乙锭染色凝胶
      3. 切割包括扩增的DNA片段(约1.6-kbp)的凝胶区域
      4. 使用illustra GFX PCR和凝胶带纯化试剂盒从凝胶纯化PCR扩增的DNA 注意:在这个过程中,我们用30μl无核酸酶的水洗脱DNA片段(8μl等分的洗脱液用作体外转录的模板DNA)。

  2. 在T7启动子控制下的荧光素酶mRNA的体外转录
    1. 准备体外转录组合。
      在室温下混合以下组分:
      8μl模板DNA
      2μl10x反应缓冲液
      2μl10mM ATP
      2μl10mM CTP
      2μl10mM UTP
      2μl10mM GTP
      2μlT7酶混合物
      用移液器彻底混匀。
    2. 在37℃,4小时孵育。
    3. 加入2μlTURBO DNase(MEGAScript的组分)消化其余模板DNA,并在37℃在块培养箱中孵育15分钟。
    4. 用氯化锂(LiCl)沉淀进行RNA提取
      1. 向体外转录产物中加入30μl不含核酸酶的水和30μlLiCl沉淀溶液。
      2. 用涡流混合器彻底混合。 在-20°C下冷却1小时。
      3. 在4℃下以15,000rpm离心15分钟
      4. 弃去上清液,并用1ml 80%乙醇洗涤沉淀
      5. 在4℃下以15,000rpm离心15分钟
      6. 弃去上清液,用离心浓缩器CC105干燥沉淀物5分钟
      7.  将RNA重悬于20μl不含核酸酶的水中,储存于-80℃。

  3. RNA 5'端的酶修饰
    1. 用ScriptCap系统将m7G帽或m7G帽和2'-O甲基化加入到体外转录的RNA中。
      1. 用无核酸酶水将转录的RNA(50μg)的体积调节至67μl
      2. 使RNA在65℃变性10分钟,然后立即将管转移至冰
      3. 当RNA变性时,混合以下组分 10μl10x ScriptCap Capping Buffer
        10μl10mM GTP
        2.5μl的20mM SAM
        2.5μlScriptGuard RNase抑制剂
        4μl用于2'-O甲基化的5'加帽的RNA或无核酸酶的2'-O未甲基化的5'加帽的RNA的ScriptCap 2'-O-甲基转移酶(100U /μl)
      4. 向步骤C1c的反应混合物中加入4μlScriptCap Capping Enzyme(10 U /μl)和67μl变性RNA。
      5. 在37℃孵育2小时
      6. 反应产物用RNeasy Mini Kit纯化
  4. 用小鼠胚胎成纤维细胞进行RNA转染
    1. 用2×10 5个小鼠胚胎成纤维细胞在1ml DMEM中接种35mm直径的培养皿,并孵育24小时。
    2. 使用Lipofectamine 2000转染RNA。
      1. 在每个样品240μlOpti-MEM培养基中稀释10μlLipofectamine 2000 DNA转染试剂。
      2. 在250μlOpti-MEM培养基中稀释2μg荧光素酶RNA
      3. 添加250微升稀释Lipofectamine 2000 DNA转染试剂到250微升稀释的荧光素酶RNA。 彻底混合。
      4. 在室温下孵育15分钟
      5. 添加RNA-脂质复合物到小鼠胚胎成纤维细胞,并孵育6小时
  5. 荧光素酶翻译记者测定
    1. 细胞裂解物的制备。
      1. 孵育6小时后,用D-PBS( - )洗涤RNA转染的小鼠胚胎成纤维细胞两次。
      2. 加入100微升1×无源裂解缓冲液,收获细胞用细胞刮刀
      3. 在4℃下以15,000rpm离心15分钟
      4. 通过BCA蛋白测定确定5μl等分的上清液中的蛋白浓度
      5. 将等量上清液的等量蛋白质(通过BCA蛋白质测定确定)加入50μl荧光素酶测定试剂II(LAR II)中。
      6.  立即,根据制造商的说明,在Lumat LB 9507发光计上测量相对荧光素酶单位(RLUs)。


        图1.引入的RNA的荧光素酶活性。野生型和 Ifit1 缺乏( Ifit1 -/- )小鼠胚胎成纤维细胞用三种不同类型的荧光素酶mRNA(5'-未封端,5'封端,但2'-O未甲基化; 5'帽+/2'-O Me-,5'封端和2'-O-甲基化; 5'帽+/2'-O Me +)。荧光素酶活性显示为相对光单位(RLU),并且RLU的数目通过步骤E1d中测定的蛋白质的浓度标准化。数据显示为一式三份样品的平均值±SD。这些数据表明Ifit1选择性抑制5'加帽的但2'-O非甲基化(5'帽+/2'-O Me - )荧光素酶mRNA的翻译。

致谢

这项工作改编自以下文件:Kimura等人(2013)。 这项工作得到教育,文化,体育,科学技术部,日本科学技术局和卫生,劳动和福利部的资助。

参考文献

  1. Kimura,T.,Katoh,H.,Kayama,H.,Saiga,H.,Okuyama,M.,Okamoto,T.,Umemoto,E.,Matsuura,Y.,Yamamoto,M.and Takeda, 2013)。 Ifit1通过绑定到5'加帽来抑制日本脑炎病毒复制 2'-O unmethylated RNA。 J Virol  87(18):9997-10003。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kimura, T. and Takeda, K. (2014). Cellular Translational Reporter Assay. Bio-protocol 4(6): e1068. DOI: 10.21769/BioProtoc.1068.
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