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This protocol is for testing responses of a candidate cell line/cell lines to Hh ligands or Hh pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Hh pathway responses. Canonical Hh signaling activity transcriptionally induces Hh target genes that contain consensus Gli binding element. Hh-responsive cells transiently or stably expressing luciferase protein under the regulation of the Gli promoter element can be used to report stimulus-dependent Hh-pathway activity.

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Hedgehog (Hh) Reporter Activity Assay
Hedgehog基因活性报告实验

发育生物学 > 细胞信号传导 > 配体
作者: Chen Zhao
Chen ZhaoAffiliation: Department of Developmental Biology, Institute for Stem Cell and Regenerative Medicine, Stanford University, Stanford, USA
For correspondence: chenzhao@stanford.edu
Bio-protocol author page: a1513
Vol 4, Iss 14, 7/20/2014, 4911 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1182

[Abstract] This protocol is for testing responses of a candidate cell line/cell lines to Hh ligands or Hh pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Hh pathway responses. Canonical Hh signaling activity transcriptionally induces Hh target genes that contain consensus Gli binding element. Hh-responsive cells transiently or stably expressing luciferase protein under the regulation of the Gli promoter element can be used to report stimulus-dependent Hh-pathway activity.

[Abstract] 该方案用于测试候选细胞系/细胞系对Hh配体或Hh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Hh通路反应的活性的小分子文库或生物制剂。 规范Hh信号活动转录诱导含有共有Gli结合元件的Hh靶基因。 在Gli启动子元件的调节下瞬时或稳定表达荧光素酶蛋白的Hh反应性细胞可用于报告刺激依赖性Hh-途径活性。

Materials and Reagents

  1. NIH3T3 cells or NIH3T3-Light2 cells (ATCC, catalog number: JHU-68 )
    Note: NIH3T3-Light2 cells were initially generated in Dr. Beachy’s Laboratory and were deposited in ATCC, the public available source of biomedical reagents including laboratory made cell lines. This cell line is available from Dr. Beachy’s lab per request.
  2. Hh-conditioned medium
  3. Fetal bovine serum (FBS)
  4. Bovine calf serum (BCS)
  5. DMEM (high glucose) (Life Technologies, InvitrogenTM, catalog number: 11965 )
  6. TransIT-2020 (Mirius, catalog number: MIR5404 )
  7. 8XGli-Firefly luciferase expression construct
    Note: The 8XGli-Firefly luciferase expression construct is made at Dr. Beachy’s Laboratory. Eight consecutive repeats of Gli responding element (8XGli) are PCR amplified and cloned into the minimal enhancer region of pGL3-Luciferase [promega] vector. Map of pGL3 is available from promega.
  8. pRL-SV40-Renilla luciferase expression construct
    Note: The pRL-SV40-Renilla luciferase expression construct is made at Dr. Beachy’s Laboratory.
  9. Dual-Luciferase @ Reporter Assay System (Promega Corporation, catalog number: E1960 )
  10. Growth media (see Recipes)
  11. Shh-conditioned media (see Recipes)
  12. Serum deprived media (see Recipes)

Equipment

  1. 15 ml thermal scientific Nunc conical tubes
  2. T75 culture flask (BD Biosciences, Falcon®, catalog number: 353135 )
  3. 24-well tissue culture plate (BD Biosciences, Falcon®, catalog number: 353047 )
  4. 96-well flat-bottom plate (Corning, Costar® , catalog number: 3915 )
  5. Centrifuge (Eppendorf, model: 8810R )
  6. Water bath
  7. CO2 incubator
  8. Berthold Luminometer (Berthold Technologies, model: Centro XS LB960 )

Procedure

  1. Grow low passage NIH3T3 or NIH3T3-light2 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.
      Note: Never let cells reach 100% confluency.
    4. The cell line should be subcultured for one or two time before performing the actual assay. Then plate the cells into 24-well plates as triplicate according to the experimental conditions (for NIH3T3 cells, plate as 5 x 104 cells/well; For NIH3T3-Light2 cells, plate as 4 x 105 cells/well).

  2. Transfection (only for NIH3T3 cells, as NIH3T3-Light stably express dual luciferase reporter constructs, if work with NIH3T3-Light2 cells, go directly to step B3).
    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 8XGli 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 ShhN-treated conditions. Relative firefly/renilla signals are normalized as fold of induction to untreated conditions.
Note: Relative Firefly Luciferase (FL)/Renilla Luciferase(RL) = Raw FL/Raw RL. Fold changes of FL/RL at Shh-stimulated condition is normalized to FL/RL at unstimulated condition.

Recipes

  1. Growth media
    For NIH3T3 cells: DMEM + 10% FBS + 1% PS
    For NIH3T3-light2 cells: DMEM + 10% BCS + 1% PS
  2. Shh-conditioned media
    Note: Shh-conditioned media is prepared by collecting supernatant of HEK293 cells stably expressing N-terminal truncated form of Shh protein.
    Shh-conditioned media is diluted in a ratio of 1:10 in DMEM + 2% FBS + 1% PS.
  3. Serum deprived media
    DMEM
    2% FBS containing various concentrations of Hh agonists or diluted Shh-conditioned media

Acknowledgments

This protocol is adapted from published work (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.

材料和试剂

  1. NIH3T3细胞或NIH3T3-Light2细胞(ATCC,目录号:JHU-68)
    注意:NIH3T3-Light2细胞最初在Beachy's博士的实验室中产生,并保藏在ATCC,生物医学试剂的公开来源,包括实验室制备的细胞系。 该细胞系可以从Dr.Berryy的实验室根据要求获得。
  2. Hh条件培养基
  3. 胎牛血清(FBS)
  4. 牛血清(BCS)
  5. DMEM(高葡萄糖)(Life Technologies,Invitrogen TM ,目录号:11965)
  6. TransIT-2020(Mirius,目录号:MIR5404)
  7. 8XGli-萤火虫荧光素酶表达构建体
    注意:8XGli-Firefly荧光素酶表达构建体在Beachy's Laboratory实验室制备。 将8个连续的Gli反应元件(8XGli)重复PCR扩增并克隆到pGL3-荧光素酶[promega]载体的最小增强子区。 promega可提供pGL3的地图。
  8. 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。
  9. Dual-Luciferase @ Reporter Assay System(Promega Corporation,目录号:E1960)
  10. 生长培养基(参见食谱)
    DMEM(高葡萄糖)(Life Technologies,Invitrogen TM ,目录号:11965)
  11. TransIT-2020(Mirius,目录号:MIR5404)
  12. 8XGli-萤火虫荧光素酶表达构建体
    注意:8XGli-Firefly荧光素酶表达构建体在Beachy's Laboratory实验室制备。 将8个连续的Gli反应元件(8XGli)重复PCR扩增并克隆到pGL3-荧光素酶[promega]载体的最小增强子区。 promega可提供pGL3的地图。
  13. 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。
  14. Dual-Luciferase @ Reporter Assay System(Promega Corporation,目录号:E1960)
  15. 生长培养基(参见食谱)
    ... 24-well tissue culture plate (BD Biosciences, Falcon®, catalog number: 353047)
  16. 96-well flat-bottom plate (Corning, Costar® , catalog number: 3915)
  17. Centrifuge (Eppendorf, model: 8810R)
  18. Water bath
  19. CO2 incubator
  20. Berthold Luminometer (Berthold Technologies, model: Centro XS LB960)

程序

  1. 从液氮库生长低通量NIH3T3或NIH3T3-light2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/孔的密度接种 在37℃,5%CO 2下培养。)
    3. 当细胞生长到60%亚汇合时分裂细胞。
      注意:切勿让细胞达到100%汇合。
    4. 细胞系应进行传代培养一或两次 进行实际测定。 然后将细胞接种到24孔板中   根据实验条件一式三份(对于NIH3T3细胞,   板作为5×10 4个细胞/孔; 对于NIH3T3-Light2细胞,平板为4×10 5个细胞/孔)。

  2. 转染(仅对于NIH3T3细胞,如NIH3T3-Light稳定表达双荧光素酶报道基因构建体,如果使用NIH3T3-Light2细胞,则直接进行步骤B3)。
    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8XGli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:更换为血清剥夺培养基。
    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 腔肠素和可以检测的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 在环境温度下的发光计)。
    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.萤火虫和海肾萤光素酶信号在未处理和ShhN处理的条件下测量。相对萤火虫/海肾信号被归一化为未处理条件的诱导倍数。
注意:相对萤火虫荧光素酶(FL)/海肾萤光素酶(RL)=原始FL /原始RL。 在未刺激条件下,在Shh刺激条件下FL/RL的倍数变化归一化为FL/RL

食谱

  1. 生长介质
    对于NIH3T3细胞:DMEM + 10%FBS + 1%PS
    对于NIH3T3-light2细胞:DMEM + 10%BCS + 1%PS
  2. 有条件的媒体
    注意:Shh条件培养基通过收集稳定表达Shh蛋白的N末端截短形式的HEK293细胞的上清液来制备。
    在DMEM + 2%FBS + 1%PS中以1:10的比例稀释Shh条件培养基。
  3. 血清剥夺了媒体
    DMEM
    2%FBS,含有各种浓度的Hh激动剂或稀释的Shh条件培养基

致谢

该协议改编自已发表的工作(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 U S A 107(30):13432-13437。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zhao, C. (2014). Hedgehog (Hh) Reporter Activity Assay. Bio-protocol 4(14): e1182. DOI: 10.21769/BioProtoc.1182; Full Text
  2. Kim, J., Lee, J. J., Kim, J., Gardner, D.and Beachy, P. A. (2010). Arsenic antagonizes the Hedgehog pathway by preventingciliary accumulation and reducing stability of the Gli2 transcriptionaleffector. Proc Natl Acad Sci U S A 107(30): 13432-13437.




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