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Generation of a Cellular Reporter for Functional BRD4 Inhibition
BRD4抑制功能细胞报告基因的生成   

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Abstract

The ubiquitously expressed bromodomain-containing protein 4 (BRD4) is an epigenetic reader, which recruits transcriptional regulatory complexes to acetylated chromatin. Because of its role in enhancing proliferation, BRD4 has become a therapeutic target in oncology, as the inhibition of this protein leads to the reduction of the growth of many tumours. Even though BRD4 is more and more studied, its mechanism of action has not been fully described yet. Therefore, we aimed at generating a cellular reporter system to monitor BRD4 inhibition. Such reporter can be potentially used in high throughput chemical and genetic screenings, in order to uncover new possible BRD4 functional pathways. The deeper understanding of the mechanism of action of BRD4 activity will certainly help in developing new therapy strategies for those cancers so called BRD4-dependent.

Keywords: Cellular chromatin reporters(细胞染色质报告基因), Epigenetic(表观遗传学), Chromatin reorganization(染色质重塑), Heterochromatinization(异染色质化), BRD4(BRD4)

Background

Research in the epigenetic field has recently highlighted the central role of BRD4 in cancer progression. BRD4 is an acetyl-lysine reader of the BET (bromodomain and extraterminal domain) family (Dey et al., 2003; Filippakopoulos et al., 2012; Wang et al., 2012) able to bind to acetylated histones at promoter and enhancer regions (Dey et al., 2003; Filippakopoulos et al., 2012; Nagarajan et al., 2014). The mechanism of action of this epigenetic reader consists in the activation of gene promoters and enhancers by recruiting several transcription factors, cofactors and RNA polymerase II (RNApol II), which results in modulating, mostly enhancing, the transcription of certain target genes. The BRD4-histone module has been described to play a key role regulating cell cycle progression (Dey et al., 2003; Wu and Chiang, 2007; Yang et al., 2008; Devaiah and Singer, 2013) and genomic structure and stability (Wu and Chiang, 2007; Floyd et al., 2013); for those reasons, BRD4 has frequently been associated with cancer development and progression (Yang et al., 2008; Zuber et al., 2011; Nagarajan et al., 2014; Wu et al., 2015).

Chromatin reporter cell lines have been already developed in order to identify modulators of position effect variegation (Tchasovnikarova et al., 2015) or to discover new chromatin-targeting compound (Johnson et al., 2008; Best et al., 2011; Wang et al., 2013). In contrast to previous approaches, we wanted to develop a protocol for the generation of a reporter cell line able to monitor the BRD4-dependent heterochromatization of a generic reporter. To achieve that, we used a common retroviral vector carrying an RFP (Red Fluorescent Protein) gene, and selected clones that integrated it in fully repressed genomic regions specifically reactivated by BRD4 inhibition. The haploid nature of the cell line used (KBM7 [Andersson et al., 1995]), makes the reporter easily amenable not only to chemical screens, but also to genetic screens. Both methods can be used for the identification of new BRD4 direct and functional partners, and results from these approaches will provide further insights into BRD4 biology.

Materials and Reagents

  1. Pipette tips
  2. 6-well plates, tissue culture treated (Corning, Costar®, catalog number: 3506 )
  3. 15 ml Falcon® conical centrifuge tube (Corning, Falcon®, catalog number: 352196 )
  4. 0.45 μm syringe filters (VWR, catalog number: 514-8021 )
  5. 24-well plates, tissue culture treated (STARLAB INTERNATIONAL, catalog number: CC7682-7524 )
  6. 96-well plates, tissue culture treated (Corning, catalog number: 3598 )
  7. Viewplate-96 black, optically clear bottom, tissue culture treated, sterile, 96-Well with lid (PerkinElmer, catalog number: 6005182 )
  8. 10 cm plates, tissue culture treated (Corning, catalog number: 430167 )
  9. 293T cell line (ATCC, catalog number: CRL-3216 )
  10. KBM7 cell line (Chronic Myeloid Leukaemia) (Horizon Discovery, catalog number: C628 )
  11. Fluorescent reporter vector (LZRS-RFP-ires-ZEO retroviral vector, a gift from S. Nijman Lab, Ludwig Cancer Research, Oxford)
  12. Packaging vector (e.g., pCMV-Gag-Pol retroviral vector, Addgene, catalog number: 14887 ; pCMV-VSV-G envelope vector, Addgene, catalog number: 8454 )
  13. DMEM media (Thermo Fisher Scientific, GibcoTM, catalog number: 41965039 )
  14. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10500064 )
  15. Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  16. Opti-MEM
  17. IMDM media (Thermo Fisher Scientific, GibcoTM, catalog number: 21980032 )
  18. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190094 )
  19. (S)-JQ1 (MedChemExpress, catalog number: HY-13030 )
  20. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )

Equipment

  1. Pipettes (Gilson)
  2. Cell culture centrifuge (Sigma Laborzentrifugen, model: 3-18K , catalog number: 10290)
  3. FACS (BD, BD Bioscience, model: BD FACSCALIBUR )
  4. FACS sorter (BD, BD Biosciences, model: BD FACSAria )
  5. Fluorescence microscope (PerkinElmer, model: HH12000000 )
  6. Cell culture hood (Thermo Fisher Scientific, model: HerasafeTM KS , catalog number: 51022515)
  7. Cell culture incubator (Eppendorf, model: Galaxy® 170 R , catalog number: CO170R-230-1000)

Procedure

  1. Virus preparation
    1. 293T cells are seeded at 200,000 cells/ml and grown until 60% confluence in DMEM plus 10% FBS in 6-well plates. When they reach such confluence rate (usually the day after), they are transfected using Lipofectamine 2000, according to manufacturer’s instructions, in order to produce the virus needed for the reporter generation (see details below). In order to get optimal virus production, it is important to use 293T cells at low passage number, and which have never been overconfluent.
    2. For the LZRS-RFP-ires-ZEO retrovirus production, 1.5 μg of LZRS-RFP-ires-ZEO retroviral vector were used in combination with 1.5 μg of packaging vectors (pCMV-Gag-Pol retroviral vector 8:1 pCMV-VSV-G envelope vector). Briefly (for 1 well), vectors are diluted and gently mixed in 250 μl of Opti-MEM. 5 μl of Lipofectamine 2000 are diluted in 250 μl of Opti-MEM and incubate for 5 min at RT (room temperature). After the 5-min incubation, the vector dilution and the Lipofectamine 2000 dilution are combined and gently mixed. This suspension is incubated for 20 min at RT and then added to the cell media drop by drop. Finally, the plate is mixed gently by rocking back and forth in order to distribute homogenously the transfection solution.
    3. Virus harvest is done at 30 and 48 h post transfection by collecting the media, centrifuging it in 15 ml Falcon conical centrifuge tubes at 1,200 x g for 5 min at RT and then filtering it through 0.45 μm syringe filters into new 15 ml Falcon conical centrifuge tubes (RT).
      Note: At this point the virus can be used for titration and cell infection (steps 2 and 3) or stored at -80 °C.
  2. Virus titration
    1. KBM7 cells are seeded in a 6-well plate in IMDM plus 10% FBS at 2 x 106 cells/ml (1 ml/well), and then infected using different ratios of media/virus. The virus is resuspended in 1 ml of IMDM plus 10% FBS. In this way, the final cell concentration is of 106 cells/ml.
    2. The ratio typically tested were: 1/100, 1/40, 1/20, 1/10, 1/4 and 1/2 (virus volume vs. cell volume).
    3. 5 μg/ml (final concentration) of Polybrene is added to each well by pipetting it in the cell media, once the virus has been added (Polybrene stock solution is 5 mg/ml).
    4. 24 and 48 h post infection cells are washed in PBS and the media is replaced with fresh aliquots (2 ml/well). Cells of each well are resuspended once in 5 ml of PBS (RT) and then centrifuged at 1,200 x g for 5 min at RT.
  3. 48 h post infection, FACS analysis is performed in order to check the percentage of infection of each well/ratio. A concentration of virus giving between 25% and 40% of cell infection (25-40% of RFP positive cells) is chosen to be used for the reporter generation (therefore ensuring that likely no more than one infection event could happen per cell). LZRS-RFP-ires-ZEO infection.
    1. KBM7 cells are seeded in 6-well plates in IMDM plus 10% FBS at 106 cells/ml (30 x 106 cells are used in total).
    2. KBM7 cells are treated with 0.5 μM (S)-JQ1 for 18 h and then infected with the LZRS-RFP-ires-ZEO retrovirus (using the virus concentration determined in step 2).
    3. Media is changed after 24 and 48 h post infection; (S)-JQ1 is kept in the culture media at 0.5 μM.
  4. First sorting (pool)
    RFP-positive cells are sorted in presence of 0.5 μM (S)-JQ1 (the whole population is sorted: cells number is at this stage approximately 120 x 106).
  5. Second sorting (single cells)
    (S)-JQ1 is removed from the media and after 30 h the RFP negative population is sorted into single cell clones. Approximately 10 96-well plates are filled; the rest of cell is frozen in IMDM media plus 10% FBS and 10% DMSO.
    Note: Usually for the sorting procedure cells must be washed at least once in 5 ml of PBS (RT), centrifuged at 1,200 x g for 5 min at RT and resuspended in 0.5 ml of PBS (maximum cell concentration allowed is 30 x 106 cell/ml); we recommend to add 10% of cell media to the resuspension in order to avoid cell-cluster formation, especially during the first sorting.
  6. Single clone amplification and reporter selection
    1. Outgrowing clones (about 10 days after step 5) are amplified and frozen in aliquots of 10 x 106 cells in IMDM media plus 10% FBS and 10% DMSO. 20,000 cells of each clone are seeded in 96-well plates (Viewplate-96 black, optically clear bottom) and treated with (S)-JQ1 for 24 h.
    2. Cells are imaged using the Operetta high-content imaging system (20x objective and non-confocal mode) (Figure 1). A clone is selected as reporter if after (S)-JQ1 treatment is able to express RFP.


      Figure 1. (S)-JQ1 treatment activates RFP expression in KBM7 reporter cells. Examples of live cell imaging pictures of KBM7 reporter cells treated with 0.5 μM of (S)-JQ1 for 24 h; equal amount of DMSO was added as control (scale bar = 100 μm).

Notes

  1. All the cells used in this protocol were grown in a cell culture incubator at 37 °C and 5% CO2.
  2. KBM7 freezing media: IMDM, 20% FCS, 10% DMSO. Cells are gradually frozen at -80 °C and then passed to liquid nitrogen.
  3. LZRS-RFP-ires-ZEO vector can be replaced with any other fluorescent reporter vector, either retro or lentiviral.
  4. (S)-JQ1 treatment can be replaced with any other epigenetic or not epigenetic drug treatment in order to generate reporters for the specific enzyme of interest.
  5. KBM7 cells can be replaced with any other cell line of interest. We have chosen this cell line because they are haploid and therefore allows unambiguous monoallelic genetic configurations.

Acknowledgments

Research in the Kubicek laboratory is supported by the Austrian Federal Ministry of Science, Research and Economy; the National Foundation for Research, Technology, and Development; the Marie Curie Career Integration Grant EPICAL; and the JDRF. Sara Sdelci acknowledges support by JDRF postdoctoral fellowship 3-PDF-2014-206-A-N ‘Reprogramming by Loss of Function’.

References

  1. Andersson, B. S., Collins, V. P., Kurzrock, R., Larkin, D. W., Childs, C., Ost, A., Cork, A., Trujillo, J. M., Freireich, E. J., Siciliano, M. J. and et al. (1995). KBM-7, a human myeloid leukemia cell line with double Philadelphia chromosomes lacking normal c-ABL and BCR transcripts. Leukemia 9(12): 2100-2108.
  2. Best, A. M., Chang, J., Dull, A. B., Beutler, J. A. and Martinez, E. D. (2011). Identification of four potential epigenetic modulators from the NCI structural diversity library using a cell-based assay. J Biomed Biotechnol 2011: 868095.
  3. Devaiah, B. N. and Singer, D. S. (2013). Two faces of BRD4: mitotic bookmark and transcriptional lynchpin. Transcription 4(1): 13-17.
  4. Dey, A., Chitsaz, F., Abbasi, A., Misteli, T. and Ozato, K. (2003). The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis. Proc Natl Acad Sci U S A 100(15): 8758-8763.
  5. Filippakopoulos, P., Picaud, S., Mangos, M., Keates, T., Lambert, J. P., Barsyte-Lovejoy, D., Felletar, I., Volkmer, R., Muller, S., Pawson, T., Gingras, A. C., Arrowsmith, C. H. and Knapp, S. (2012). Histone recognition and large-scale structural analysis of the human bromodomain family. Cell 149(1): 214-231.
  6. Floyd, S. R., Pacold, M. E., Huang, Q., Clarke, S. M., Lam, F. C., Cannell, I. G., Bryson, B. D., Rameseder, J., Lee, M. J., Blake, E. J., Fydrych, A., Ho, R., Greenberger, B. A., Chen, G. C., Maffa, A., Del Rosario, A. M., Root, D. E., Carpenter, A. E., Hahn, W. C., Sabatini, D. M., Chen, C. C., White, F. M., Bradner, J. E. and Yaffe, M. B. (2013). The bromodomain protein Brd4 insulates chromatin from DNA damage signalling. Nature 498(7453): 246-250.
  7. Johnson, R. L., Huang, W., Jadhav, A., Austin, C. P., Inglese, J. and Martinez, E. D. (2008). A quantitative high-throughput screen identifies potential epigenetic modulators of gene expression. Anal Biochem 375(2): 237-248.
  8. Nagarajan, S., Hossan, T., Alawi, M., Najafova, Z., Indenbirken, D., Bedi, U., Taipaleenmaki, H., Ben-Batalla, I., Scheller, M., Loges, S., Knapp, S., Hesse, E., Chiang, C. M., Grundhoff, A. and Johnsen, S. A. (2014). Bromodomain protein BRD4 is required for estrogen receptor-dependent enhancer activation and gene transcription. Cell Rep 8(2): 460-469.
  9. Tchasovnikarova, I. A., Timms, R. T., Matheson, N. J., Wals, K., Antrobus, R., Gottgens, B., Dougan, G., Dawson, M. A. and Lehner, P. J. (2015). GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells. Science 348(6242): 1481-1485.
  10. Wang, L., Chang, J., Varghese, D., Dellinger, M., Kumar, S., Best, A. M., Ruiz, J., Bruick, R., Pena-Llopis, S., Xu, J., Babinski, D. J., Frantz, D. E., Brekken, R. A., Quinn, A. M., Simeonov, A., Easmon, J. and Martinez, E. D. (2013). A small molecule modulates Jumonji histone demethylase activity and selectively inhibits cancer growth. Nat Commun 4: 2035.
  11. Wang, R., Li, Q., Helfer, C. M., Jiao, J. and You, J. (2012). Bromodomain protein Brd4 associated with acetylated chromatin is important for maintenance of higher-order chromatin structure. J Biol Chem 287(14): 10738-10752.
  12. Wu, S. Y. and Chiang, C. M. (2007). The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation. J Biol Chem 282(18): 13141-13145.
  13. Wu, T., Pinto, H. B., Kamikawa, Y. F. and Donohoe, M. E. (2015). The BET family member BRD4 interacts with OCT4 and regulates pluripotency gene expression. Stem Cell Reports 4(3): 390-403.
  14. Yang, Z., He, N. and Zhou, Q. (2008). Brd4 recruits P-TEFb to chromosomes at late mitosis to promote G1 gene expression and cell cycle progression. Mol Cell Biol 28(3): 967-976.
  15. Zuber, J., Shi, J., Wang, E., Rappaport, A. R., Herrmann, H., Sison, E. A., Magoon, D., Qi, J., Blatt, K., Wunderlich, M., Taylor, M. J., Johns, C., Chicas, A., Mulloy, J. C., Kogan, S. C., Brown, P., Valent, P., Bradner, J. E., Lowe, S. W. and Vakoc, C. R. (2011). RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 478(7370): 524-528.

简介

普遍表达的含溴结构域的蛋白质4(BRD4)是表观遗传学读者,其将转录调节复合物募集到乙酰化染色质。 由于其在增强增殖中的作用,BRD4已经成为肿瘤学的治疗靶点,因为抑制这种蛋白质导致许多肿瘤的生长减少。 即使BRD4越来越多的研究,其行动机制尚未得到充分的描述。 因此,我们旨在产生细胞报告系统来监测BRD4抑制。 这种记录可以潜在地用于高通量化学和遗传筛选,以揭示新的可能的BRD4功能途径。 对BRD4活动作用机制的深入了解肯定有助于为所谓的BRD4依赖型癌症开发新的治疗策略。
【背景】表观遗传学领域的研究最近突出了BRD4在癌症进展中的中心作用。 BRD4是BET(溴结构域和终末外结构域)家族(Dey等人,2003; Filippakopoulos等人,2012; Wang)的乙酰赖氨酸阅读器, et al。,2012)能够结合启动子和增强子区域上的乙酰化组蛋白(Dey等人,2003; Filippakopoulos等人,2012; Nagarajan等人,,2014)。这种表观遗传学读者的作用机制在于通过招募几种转录因子,辅因子和RNA聚合酶II(RNApol II)来激活基因启动子和增强子,其导致调节,大部分增强某些靶基因的转录。已经描述了BRD4组蛋白模块发挥调控细胞周期进程的关键作用(Dey等人,2003; Wu和Chiang,2007; Yang等人, 2008; Devaiah和Singer,2013)和基因组结构和稳定性(Wu和Chiang,2007; Floyd等人,2013);由于这些原因,BRD4经常与癌症发展和进展相关(Yang等人,2008; Zuber等人,2011; Nagarajan等人,2014; Wu等人,2015)。
;已经开发了染色质报道细胞系,以便鉴定位置效应杂色的调节剂(Tchasovnikarova等人,2015)或发现新的染色质靶向化合物(Johnson等人, 2008; Best 等人,2011; Wang等人,2013)。与以前的方法相比,我们希望开发一种能够生成能够监测通用记者BRD4依赖性异染色化的报道细胞系的方案。为了实现这一点,我们使用携带RFP(红色荧光蛋白)基因的常见的逆转录病毒载体,以及将其整合到通过BRD4抑制特异性重新激活的完全受抑制的基因组区域中的选择克隆。使用的细胞系的单倍体性质(KBM7 [Andersson等人,1995])使得报告人不仅易于化学筛选,而且还适用于遗传筛选。这两种方法都可用于鉴定新的BRD4直接和功能性合作伙伴,这些方法的结果将为BRD4生物学提供进一步的见解。

关键字:细胞染色质报告基因, 表观遗传学, 染色质重塑, 异染色质化, BRD4

材料和试剂

  1. 移液器提示
  2. 6孔板,组织培养处理(Corning,Costar ®,目录号:3506)
  3. 15 ml Falcon ®锥形离心管(Corning,Falcon ®,目录号:352196)
  4. 0.45μm注射器过滤器(VWR,目录号:514-8021)
  5. 24孔板,组织培养处理(STARLAB INTERNATIONAL,目录号:CC7682-7524)
  6. 96孔板,组织培养处理(Corning,目录号:3598)
  7. 观察板-96黑色,光学透明底部,组织培养处理,无菌,96孔带盖(PerkinElmer,目录号:6005182)
  8. 10厘米平板,组织培养处理(康宁,目录号:430167)
  9. 293T细胞系(ATCC,目录号:CRL-3216)
  10. KBM7细胞系(慢性骨髓性白血病)(Horizon Discovery,目录号:C628)
  11. 荧光报道载体(LZRS-RFP-ires-ZEO逆转录病毒载体,来自S.Nijman Lab,Ludwig Cancer Research,Oxford)的一份礼物
  12. 包装载体(例如,pCMV-Gag-Pol逆转录病毒载体,Addgene,目录号:14887; pCMV-VSV-G包膜载体,Addgene,目录号:8454)
  13. DMEM培养基(Thermo Fisher Scientific,Gibco TM,目录号:41965039)
  14. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10500064)
  15. Lipofectamine 2000(Thermo Fisher Scientific,Invitrogen TM,目录号:11668019)
  16. Opti-MEM
  17. IMDM介质(Thermo Fisher Scientific,Gibco TM ,目录号:21980032)
  18. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190094)
  19. S ) - JQ1(MedChemExpress,目录号:HY-13030)
  20. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)

设备

  1. 移液器(Gilson)
  2. 细胞培养离心机(Sigma Laborzentrifugen,型号:3-18K,目录号:10290)
  3. FACS(BD,BD Bioscience,型号:BD FACSCALIBUR)
  4. FACS分选机(BD,BD Biosciences,型号:BD FACSAria)
  5. 荧光显微镜(PerkinElmer,型号:HH12000000)
  6. 细胞培养罩(Thermo Fisher Scientific,型号:Herasafe TM,目录号:51022515)
  7. 细胞培养培养箱(Eppendorf,型号:Galaxy 170R,目录号:CO170R-230-1000)

程序

  1. 病毒准备
    1. 将293T细胞以20万个细胞/ ml接种,并在6孔板中在DMEM加10%FBS中生长至60%汇合。当它们达到这样的汇合率(通常是在一天之后)时,根据制造商的说明书,它们使用Lipofectamine 2000转染,以产生报告生成所需的病毒(见下文)。为了获得最佳的病毒生产,重要的是使用低通道数的293T细胞,并且从未过度融合。
    2. 对于LZRS-RFP-ires-ZEO逆转录病毒生产,将1.5μgLZRS-RFP-ires-ZEO逆转录病毒载体与1.5μg包装载体(pCMV-Gag-Pol逆转录病毒载体8:1 pCMV-VSV-G包络矢量)。简单地(1孔),将载体稀释并轻轻混合在250μlOpti-MEM中。将5μl的Lipofectamine 2000稀释在250μl的Opti-MEM中,并在RT(室温)下孵育5分钟。孵育5分钟后,将载体稀释液和Lipofectamine 2000稀释液合并并轻轻混合。将该悬浮液在室温下孵育20分钟,然后逐滴加入细胞培养基中。最后,通过前后摆动轻轻地混合板,以均匀分布转染溶液。
    3. 在转染后30和48小时通过收集培养基进行病毒收获,在室温下将其在1500ml Falcon圆锥形离心管中离心15分钟,然后通过0.45μm注射器过滤器过滤新的15 ml Falcon锥形离心管(RT)。
      注意:此时病毒可用于滴定和细胞感染(步骤2和3)或存储于-80°C。
  2. 病毒滴定
    1. 将KBM7细胞以2×10 6细胞/ ml(1ml /孔)的IMDM加10%FBS的6孔板接种,然后使用不同比例的培养基/病毒进行感染。将病毒重新悬浮于1ml IMDM加10%FBS中。以这种方式,最终细胞浓度为10μg/ ml细胞/ ml
    2. 通常测试的比例是:1/100,1/40,1/20,1/10,1/4和1/2(病毒体积对细胞体积)。
    3. 一旦加入病毒(Polybrene储备溶液为5 mg / ml),将细胞培养基中的每个孔加入5μg/ ml(终浓度)的聚凝胺。
    4. 感染后24和48小时,细胞在PBS中洗涤,培养基用新鲜等分试样(2ml /孔)置换。每孔的细胞在5ml PBS(RT)中重新悬浮一次,然后在室温下以1,200×g离心5分钟。
  3. 感染后48小时,进行FACS分析,以检查每个井/感染率的百分比。选择使细胞感染的25%至40%的病毒浓度(25-40%的RFP阳性细胞)用于记者生成(因此确保每个细胞可能不会发生多于一次的感染事件)。 LZRS-RFP-ires-ZEO感染。
    1. 将KBM7细胞接种在10-μl细胞/ ml(总共30×10 6个细胞)的IMDM加10%FBS的6孔板中。
    2. 将KBM7细胞用0.5μM(J)JQ1处理18小时,然后用LZRS-RFP-ires-ZEO逆转录病毒感染(使用在步骤2中测定的病毒浓度)。
    3. 感染后24和48 h培养基发生变化; ( S ) - JQ1以0.5μM保存在培养基中
  4. 首先排序(池)
    RFP阳性细胞在0.5μM(SEQ ID NO:)JQ1存在下分选(整个群体分类:细胞数目在此阶段约120×10 -6)。
  5. 二次排序(单细胞)
    () - JQ1从培养基中除去,30小时后将RFP阴性人群分选成单细胞克隆。填充约10个96孔板;其余细胞在IMDM培养基中加10%FBS和10%DMSO冷冻 注意:通常对于分选过程,细胞必须在5ml PBS(RT)中洗涤至少一次,在室温下以1,200×g离心5分钟,并重悬于0.5ml PBS中(允许的最大细胞浓度为30x 10
  6. 细胞/ ml);我们建议将10%的细胞培养基加入重悬,以避免细胞簇形成,特别是在首次分选过程中。
  7. 单克隆扩增和记录选择
    1. 在IMDM培养基加10%FBS和10%DMSO的10×10 6个细胞的等分试样中扩增出大量的克隆(步骤5后约10天)。将每个克隆的20,000个细胞接种在96孔板(Viewplate-96黑色,光学透明的底部)中,并用(J) - JQ1处理24小时。
    2. 使用Operetta高内容成像系统(20x物镜和非共焦模式)对图像进行成像(图1)。如果之后() - JQ1治疗能够表达RFP,则选择克隆作为报告者。


      图1
    3. S - JQ1治疗激活KBM7报告细胞中的RFP表达。活细胞成像用0.5μM()JQ1处理24小时的KBM7报道细胞的图片;加入等量的DMSO作为对照(比例尺=100μm)

笔记

  1. 在该方案中使用的所有细胞在37℃和5%CO 2的细胞培养培养箱中生长。
  2. KBM7冷冻介质:IMDM,20%FCS,10%DMSO。细胞在-80℃逐渐冷冻,然后送至液氮
  3. LZRS-RFP-ires-ZEO载体可以用任何其他荧光报告载体替代,复制型或慢病毒载体。
  4. () - JQ1治疗可以用任何其他表观遗传学或非表观遗传学药物治疗来替代,以便为感兴趣的特定酶产生报告。
  5. KBM7细胞可以用任何其他感兴趣的细胞系替代。我们选择了这种细胞系,因为它们是单倍体的,因此允许明确的单等位基因配置

致谢

科比切克实验室的研究得到了奥地利联邦科学和经济部的支持。国家科技研究发展基金会;玛丽居里职业融合奖励EPICAL;和JDRF。 Sara Sdelci承认JDRF博士后研究金3-PDF-2014-206-A-N“功能丧失重编程”的支持。

参考

  1. Andersson,B.S.,Collins,V.P.,Kurzrock,R.,Larkin,D.W.,Childs,C.,Ost,A.,Cork,A.,Trujillo,J.M.,Freireich,E.J。,Siciliano,M.J。 (1995年)。 KBM-7,一种具有双重费城染色体的人类骨髓性白血病细胞系缺乏正常的c-ABL和BCR转录本。 白血病 9(12):2100-2108。
  2. Best,AM,Chang,J.,Dull,AB,Beutler,JA和Martinez,ED(2011)。&nbsp; 使用基于细胞的测定法鉴定来自NCI结构多样性库的四种潜在的表观遗传调节剂。生物生物技术 2011:868095. < br />
  3. Devaiah,BN and Singer,DS(2013)。&nbsp; Two BRD4的面孔:有丝分裂书签和转录小鼠。 转录 4(1):13-17。
  4. Dey,A.,Chitsaz,F.,Abbasi,A.,Misteli,T.和Ozato,K。(2003)。双溴结构域蛋白Brd4在间期和有丝分裂期间与乙酰化的染色质结合。 Proc Natl Acad Sci USA 100(15) :8758-8763。
  5. Filippakopoulos,P.,Picaud,S.,Mangos,M.,Keates,T.,Lambert,JP,Barsyte-Lovejoy,D.,Felletar,I.,Volkmer,R.,Muller,S.,Pawson, ,Gingras,AC,Arrowsmith,CH和Knapp,S。(2012)。人类溴结构域家族的组蛋白识别和大规模结构分析。
  6. 细胞 149(1):214-231。
  7. Floyd,SR,Pacold,ME,Huang,Q.,Clarke,SM,Lam,FC,Cannell,IG,Bryson,BD,Rameseder,J.,Lee,MJ,Blake,EJ,Fydrych,A.,Ho,R ,Greenberger,BA,Chen,GC,Maffa,A.,Del Rosario,AM,Root,DE,Carpenter,AE,Hahn,WC,Sabatini,DM,Chen,CC,White,FM,Bradner,JE和Yaffe, MB(2013)。溴结构域蛋白Brd4将染色质从DNA损伤信号。自然 498(7453):246-250。
  8. Johnson,RL,Huang,W.,Jadhav,A.,Austin,CP,Inglese,J.and Martinez,ED(2008)。&lt; a class =“ke-insertfile”href =“http: ncbi.nlm.nih.gov/pubmed/18211814“target =”_ blank“>定量高通量屏幕识别基因表达的潜在表观遗传调节剂。 375(2): 237-248。
  9. Nagarajan,S.,Hossan,T.,Alawi,M.,Najafova,Z.,Indenbirken,D.,Bedi,U.,Taipaleenmaki,H.,Ben-Batalla,I.,Scheller,M.,Loges,S 。,Knapp,S.,Hesse,E.,Chiang,CM,Grundhoff,A.and Johnsen,SA(2014)。&lt; a class =“ke-insertfile”href =“http://www.ncbi。 nlm.nih.gov/pubmed/25017071“target =”_ blank“> Bromodomain蛋白BRD4是雌激素受体依赖性增强子激活和基因转录所必需的。 8(2): 460-469。
  10. Tchasovnikarova,IA,Timms,RT,Matheson,NJ,Wals,K.,Antrobus,R.,Gottgens,B.,Dougan,G.,Dawson,MA和Lehner,PJ(2015)。&lt; a class = ke-insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/26022416“target =”_ blank“>基因沉默。 HUSH复合物的表观遗传学沉默介导人类细胞中的位置效应杂色。 348(6242):1481-1485。
  11. Wang,L.,Chang,J.,Varghese,D.,Dellinger,M.,Kumar,S.,Best,AM,Ruiz,J.,Bruick,R.,Pena-Llopis,S.,Xu,J. ,Babinski,DJ,Frantz,DE,Brekken,RA,Quinn,AM,Simeonov,A.,Easmon,J.and Martinez,ED(2013)。&nbsp; 一个小分子调节Jumonji组蛋白脱甲基酶活性,并选择性抑制癌症生长。 Nat Commun 4 :2035.
  12. Wang,R.,Li,Q.,Helfer,C.M.,Jiao,J.and You,J.(2012)。 与乙酰化染色质相关的溴霉素蛋白Brd4对于维持较高级别的染色体是重要的,订单染色质结构。 J Biol Chem 287(14):10738-10752。
  13. Wu,SY and Chiang,CM(2007)。&nbsp; 含双溴结构域的染色质接头Brd4和转录调控。生物化学282(18):13141-13145。
  14. Wu,T.,Pinto,HB,Kamikawa,YF and Donohoe,ME(2015)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/ 25684227“target =”_ blank“> BET家族成员BRD4与OCT4相互作用并调节多能性基因表达。
  15. 干细胞报告 4(3):390-403。
  16. Yang,Z.,He,N. and Zhou,Q.(2008)。&nbsp; Brd4在有丝分裂晚期向染色体招募P-TEFb以促进G1基因表达和细胞周期进程。细胞生物学 28(3):967-976。 br />
  17. Zuber,J.,Shi,J.,Wang,E.,Rappaport,AR,Herrmann,H.,Sison,EA,Magoon,D.,Qi,J.,Blatt,K.,Wunderlich,M.,Taylor, MJ,Johns,C.,Chicas,A.,Mulloy,JC,Kogan,SC,Brown,P.,Valent,P.,Bradner,JE,Lowe,SW and Vakoc,CR(2011) =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/21814200”target =“_ blank”> RNAi筛选鉴定Brd4作为急性骨髓性白血病的治疗靶点。自然 478(7370):524-528。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sdelci, S. and Kubicek, S. (2017). Generation of a Cellular Reporter for Functional BRD4 Inhibition. Bio-protocol 7(13): e2368. DOI: 10.21769/BioProtoc.2368.
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