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Wnt Reporter Activity Assay
Wnt基因活性报告实验   

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Abstract

This protocol is for testing responses of a candidate cell line/cell lines to Wnt ligands or Wnt pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Wnt pathway responses. Canonical Wnt signaling activity transcriptionally induces Wnt target genes that contain concensus TCF/LEF binding element. Wnt pathway activity responsive cells transiently or stably expressing luciferase proteins under the TCF/LEF promoter element can be used to report stimulus-dependent Wnt-pathway activity. We acquired the TopFlash (TCL/LEF-Firefly luciferase) construct from Addgene.

Materials and Reagents

  1. NIH3T3 cells or HEK293 cells
  2. Purified active Wnt3a proteins (ATCC, catalog number: CRL­2647 TM)
  3. Fetal bovine serum (FBS)
  4. DMEM (high glucose) (Life Technologies, InvitrogenTM, catalog number: 11965 )
  5. TransIT-2020 (Mirus Bio LLC, catalog number: MIR5404 )
  6. TCL/LEF-Firefly luciferase expression construct (Addgene, catalog number: 12456 )
  7. pRL-SV40-Renilla luciferase expression construct
    Note: The pRL-SV40-Renilla luciferase expression construct is made at Dr. Beachy’s Laboratory.
  8. Dual-Luciferase @ Reporter Assay System (Promega Corporation, catalog number: E1960 )
  9. Growth media (see Recipes)
  10. Wnt3a-conditioned media (see Recipes)
  11. Serum-deprived media (see Recipes)

Equipment

  1. T75 culture flask (BD Biosciences, Falcon®, catalog number: 353135 )
  2. 24-well tissue culture plate (BD Biosciences, Falcon®, catalog number: 353047 )
  3. 96-well flat-bottom plate (Corning, Costar®, catalog number: 3915 )
  4. Centrifuge (Eppendorf, model: 8810R )
  5. Water bath 
  6. CO2 incubator
  7. Berthold Luminometer (Berthold Tecnologies, model: Centro XS LB960 )

Procedure

  1. Grow low passage NIH3T3 or HEK293 cells from liquid nitrogen stock
    1. Briefly thaw cells at 37 °C water bath.
    2. Pipette cells into 15 ml thermal scientific Nunc conical tubes and spin for 5 min at 1,500 x g. Aspirate media, re-suspend cells in 15 ml growth media and plate in T75 culture flask (Cells are plated at the density at 1 x 106  and are cultured at 37 °C with 5% CO2.).
    3. Split the cells when they grow to 60% sub-confluency.
    4. The cell line should be subculturing for one or two time before performing the actual assay, and then plate the cells into 24-well plates as triplicate according to the experimental conditions (5 x 104 cells/well).

  2. Transfection
    1. Day 0: Seed cells (density as above-mentioned) in a total volume of 500 μl complete growth media (DMEM/10% FBS).
    2. Day 1: Transfection (use ratio of 7XTCL/LEF-firefly luciferase: pRL-SV40-Renilla luciferase as 10: 1; use 1.5 μl TransIT-2020/300 ng of DNA/well).
    3. Day 2: Change media 24 h after transfection (optimal).
    4. Day 3: Change to serum-deprived media.
    5. Day 5: Lyse cells (1x whole cell lysis buffer available from the Dual-Luciferase @ Reporter Assay System) and collect aliquot of supernatant (20 μl) from cell lysis, plate them into 96-well plate. 

  3. Reading luminescence signal
    1. Thaw dual-luciferase reporter reagents.
    2. Flash and prime Berthold luminometer with firefly luciferase and renilla luciferase substrate reagents. Firefly luciferase substrate is a derivative of D-luciferin. In response to D-luciferin enzymatic activity (firefly luciferase), a firefly luciferase chemical reaction will generate oxyluciferin and a specific light signal at 560 nm that can be detected by the luminometer. Renilla luciferase substrate is a derivative of Coelenterazine. In response to renilla enzymatic activity (renilla luciferase), a renilla luciferase chemical reaction will generate coelenteramide and a specific light signal at 480 nm that can be detected by the luminometer. 20 μl of each substrate is added sequencially by the luminometer and light signals generated are instantly measured by the luminometer (Light signals are detected at an enclosed chamber of the luminometer at ambient temperature).
    3. Read firefly and renilla luciferase signals (firefly luciferase signal is detected at 560 nm and renilla luciferase signal is detected at 480 nm).

Representative data



Figure 1. Firefly and Renilla luciferase signals are measured at untreated and Wnt3a treated conditions. Relative firefly/renilla signals are normalized as fold of induction to untreated conditions. Relative Firefly Luciferase (FL)/Renilla Luciferase(RL) = Raw FL/Raw RL
Note: Fold changes of FL/RL at Wnt3a-stimulated condition is normalized to FL/RL at unstimulated condition.

Recipes

  1. Growth media
    For NIH3T3 and HEK cells: DMEM + 10% FBS +1% PS
  2. Wnt3a-conditioned media
    Wnt3a-conditioned media are prepared by collecting supernatant of mouse fibroblast cells stably expressing L-Wnt3a protein, and then diluted in a ratio of 1: 10 in DMEM + 2% FBS + 1% PS.
  3. Serum-deprived media
    DMEM
    2% FBS containing various concentrations of Wnt3a-conditioned media

Acknowledgments

This protocol is adapted from Kim et al. (2010). I thank the current and past members of the Beachy lab, Stanford University, who contributed to the development of this protocol. I acknowledge the Susan G. Komen for the Cure Postdoctoral Fellowship: KG111253.

References

  1. Kim, J., Lee, J. J., Kim, J., Gardner, D. and Beachy, P. A. (2010). Arsenic antagonizes the Hedgehog pathway by preventing ciliary accumulation and reducing stability of the Gli2 transcriptional effector. Proc Natl Acad Sci U S A 107(30): 13432-13437.

简介

该方案用于测试候选细胞系/细胞系对Wnt配体或Wnt通路激动剂刺激的反应。 该方案还可以适于筛选含有增加或降低Wnt途径反应的活性的小分子文库或生物制品。 规范Wnt信号活动转录诱导含有概括TCF/LEF结合元件的Wnt靶基因。 在TCF/LEF启动子元件下瞬时或稳定表达荧光素酶蛋白的Wnt途径活性应答性细胞可用于报告刺激依赖性Wnt通路活性。 我们从Addgene获得TopFlash(TCL/LEF-Firefly萤光素酶)构建体。

材料和试剂

  1. NIH3T3细胞或HEK293细胞
  2. 纯化的活性Wnt3a蛋白(ATCC,目录号:CRL2647TM)
  3. 胎牛血清(FBS)
  4. DMEM(高葡萄糖)(Life Technologies,Invitrogen TM ,目录号:11965)
  5. TransIT-2020(Mirus Bio LLC,目录号:MIR5404)
  6. TCL/LEF-萤火虫荧光素酶表达构建体(Addgene,目录号:12456)
  7. pRL-SV40-海肾萤光素酶表达构建体
    注意:pRL-SV40-Renilla荧光素酶表达构建体是在Beachy's博士实验室。
  8. Dual-Luciferase @ Reporter Assay System(Promega Corporation,目录号:E1960)
  9. 生长培养基(参见食谱)
  10. Wnt3a条件培养基(参见配方)
  11. 血清剥夺的媒体(见配方)

设备

  1. T75培养瓶(BD Biosciences,Falcon ,目录号:353135)
  2. 24孔组织培养板(BD Biosciences,Falcon ,目录号:353047)
  3. 96孔平底板(Corning,Costar ,目录号:3915)
  4. 离心机(Eppendorf,型号:8810R)
  5. 水浴
  6. CO <2>孵化器
  7. Berthold照度计(Berthold Tecnologies,型号:Centro XS LB960)

程序

  1. 从液氮库生长低通量NIH3T3或HEK293细胞
    1. 在37℃水浴中简单解冻细胞
    2. 吸管细胞成15 ml热科学Nunc锥形管中,并在1,500x下旋转5分钟 g 。 吸出培养基,将细胞重悬在15ml生长培养基中并平板 T75培养瓶(细胞以1×10 6个细胞/孔的密度铺板) 在37℃,5%CO 2下培养)
    3. 当细胞生长到60%亚汇合时分裂细胞。
    4. 细胞系应进行传代培养一或两次 进行实际测定,然后将细胞平板到24孔 平板,根据实验条件(5×10 4个细胞/孔)一式三份。

  2. 转染
    1. 第0天:在总体积为500μl完全生长培养基(DMEM/10%FBS)中的种子细胞(如上所述的密度)。
    2. 第1天:转染(使用比例为7XTCL/LEF-萤火虫荧光素酶: pRL-SV40-海肾荧光素酶为10:1; 使用1.5μlTransIT-2020/300 ng DNA/well)
    3. 第2天:转染后24小时更换培养基(最佳)
    4. 第3天:更换为血清贫乏的培养基。
    5. 第5天:裂解细胞(1×全细胞裂解缓冲液, 双荧光素酶@报道测定系统)并收集等分试样 来自细胞裂解的上清液(20μl),将它们平板接种到96孔板中。

  3. 读取发光信号
    1. 解冻双荧光素酶报道试剂。
    2. 闪光和素数 Berthold发光计与萤火虫荧光素酶和海肾荧光素酶 底物试剂。 萤火虫荧光素酶底物是的衍生物 D-荧光素。 响应D-荧光素酶活性(萤火虫 荧光素酶),将产生萤火虫荧光素酶化学反应 氧化荧光素和可以检测的560nm处的特定光信号 通过发光计。 海肾荧光素酶底物是的衍生物 腔肠素。 响应海肾酶活性(海肾 荧光素酶),会产生海肾荧光素酶化学反应 腔肠酰胺和可以检测的480nm的特定光信号   通过发光计。 向每个底物中顺序加入20μl 所产生的光度计和光信号被立即测量 发光计(在封闭室中检测光信号 照度计在环境温度下)
    3. 阅读萤火虫和 海肾萤光素酶信号(萤火虫荧光素酶信号在 在480nm检测海肾荧光素酶信号)。

代表数据



图1.在未处理和Wnt3a处理的条件下测量萤火虫和海肾萤光素酶信号。相对萤火虫/海肾信号被归一化为未处理条件的诱导倍数。 相对萤火虫荧光素酶(FL)/Renilla荧光素酶(RL)=原始FL/Raw RL
注意:在Wnt3a刺激条件下FL/RL的倍数变化在未刺激条件下归一化为FL/RL。

食谱

  1. 生长介质
    对于NIH3T3和HEK细胞:DMEM + 10%FBS + 1%PS
  2. Wnt3a条件培养基
    通过收集稳定表达L-Wnt3a蛋白的小鼠成纤维细胞的上清液,然后以1:10的比例在DMEM + 2%FBS + 1%PS中稀释来制备Wnt3a条件培养基。
  3. 血清剥夺的媒体
    DMEM
    2%FBS,含有各种浓度的Wnt3a条件培养基

致谢

该协议改编自Kim等人(2010)。 我感谢当前和过去的沙滩实验室,斯坦福大学的成员,他们为这个协议的发展做出了贡献。 我承认苏珊G. Komen治愈博士后研究金:KG111253。

参考文献

  1. Kim,J.,Lee,J.J.,Kim,J.,Gardner,D.and Beachy,P.A。(2010)。 砷通过阻止睫状体积聚和降低Gli2转录效应物的稳定性来拮抗Hedgehog通路。 Proc Natl Acad Sci USA 107(30):13432-13437。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhao, C. (2014). Wnt Reporter Activity Assay. Bio-protocol 4(14): e1183. DOI: 10.21769/BioProtoc.1183.
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Kathryn Kilpatrick
University of Colorado- Denver
Am I missing where you are using the Wnt3a conditioned media? I added it to my 293t-OT cells and they are not fluorescing at all.
9/8/2017 8:42:53 AM Reply