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Nov 2013

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Novel Method for Site-specific Induction of Oxidative DNA Damage to Study Recruitment of Repair Proteins to Heterochromatin and Euchromatin
研究异染色体和常染色质修复蛋白补充的特定位点诱导DNA氧化损伤新方法   

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Abstract

ROS-induced DNA damage is repaired in living cells within a temporal and spatial context, and chromatin structure is critical to a consideration of DNA repair processes in situ. It’s well known that chromatin remodeling factors participate in many DNA damage repair pathways, indicating the importance of chromatin remodeling in facilitating DNA damage repair. To date, there has been no method to induce site-specific oxidative DNA damage in living cells. Therefore, it is not known whether the DNA repair mechanisms differ within active or condensed chromatin. We recently established a novel method, DTG (Damage Targeted at one Genome-site), to study DNA damage response of reactive oxygen species (ROS)-induced DNA damage in living cell at one genome loci with active or inactive transcription. For this, we integrated a tetracycline responsive elements (TRE) cassette (~90 kb) at X-chromosome in U2OS cells (Lan et al., 2010), then fused KillerRed (KR), a light-stimulated ROS-inducer which can specifically produce ROS-induced DNA damage, to a tet-repressor (tetR-KR, OFF) or a transcription activator (TA-KR, ON) (Lan et al., 2014) (Figure 1). TetR-KR or TA-KR binds to the TRE cassette and induces ROS damage under hetero- or euchromatin states, respectively. How chromatin states regulate the DNA damage response processes can be examined by using this powerful method.



Figure 1. Scheme of the DTG system. A. Scheme of tetR and TA tagged KR expression in the U2OS TRE cell line. To induce ROS-mediated damage at a specific locus in the genome, we fused KR to the tetracycline repressor to induce ROS damage in a 90 kb tetracycline response element (TRE) array (totally 96 repeats) in U2OS cells. B. Expression of tetR-KR in U2OS TRE cell line.

Materials and Reagents

  1. U2OS-SCE cell line (made in our laboratory) (Lan et al., 2010)
    Notes:
    1. In this cell line, 200 copies of pTRE / I-SceI were integrated in U2OS cells.
    2. Cells are cultured with Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (EuroClone S.p.A. P.IVA, catalog number: ECM0103L ) with 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F9665 ).
    3. For preparation of cells, cells were washed with PBS without Ca2+ and Mg2+ (EuroClone S.p.A. P.IVA, catalog number: ECB4004L ), trypsinized with Trypsin/EDTA without out Ca2+ and Mg2+ (Thermo Fisher Scientific, catalog number: BW17161E ).
  2. Plasmids
    1. pBROAD3/tetR-KR
    2. pBROAD3/tetR-mCherry
    3. pBROAD3/TA-KR 
    4. pBROAD3/TA-mCherry
      Note: pBROAD3/tetR-mcherry was provided by Dr. Edith Heard (Masui et al., 2011); pBROAD3/tetR-KR, pBROAD3/TA-mCherry, pBROAD3/TA-KR were made in our laboratory (Lan et al., 2014). Sequences are available based on requests.
  3. GFP-fusion protein or endogenous protein
  4. Lipofectamine 2000 (Life Technologies, catalog number: 12566-014 )
  5. Opti-MEM (Life Technologies, catalog number: 51985-091 )
  6. 35 mm glass bottom culture dishes (MatTek, catalog number: P35GC-1.5-14-C )
  7. IMMOIL-F30CC (Chip Humphries, catalog number: Z-81225 )

Equipment

  1. 35 mm glass bottom culture dishes
  2. 37 °C, 5% CO2 cell culture incubator
  3. Olympus FV1000 confocal microscopy system (OLYMPUS, model: FV1000- FILTER DETECT ; SYS.PACKAGE: IX81-1 405/M_AR/559/635N) with 488, 559 nm lasers
  4. PLAPON 60x oil lens (super chromatic abe. corr. obj W/1.4NA FV) (OLYMPUS, catalog number: FM1-U2B990 )
  5. Thermo-plate (MATS-U52RA26 for IX81/71/51/70/50, metal insert, HQ control) (OLYMPUS, catalog number: OTH-I0126 )
  6. 15 watt cool white fluorescent bulb (OSRAM SYLVANIA)
  7. A stage UVP (Upland, CA)

Procedure

  1. Seed U2OS-SCE cells into 35 mm glass bottom culture dishes at an approximate density of 5 x 105 per dish in DMEM with 10% FBS at 37 °C incubator with 5% CO2 for overnight.
  2. For GFP-fusion protein, co-transfect 1 µg GFP-Fusion protein with any 1 µg of pBROAD3/tetR-KR, pBROAD3/tetR-mCherry, pBROAD3/TA-KR and pBROAD3/TA-mCherry using Lipofectamine 2000. For endogenous protein, just transfect anyone of pBROAD3/tetR-KR, pBROAD3/tetR-mCherry, pBROAD3/TA-KR and pBROAD3/TA-mCherry. Follow the transfection protocol of Lipofectamine 2000.
  3. Then incubate in the 5% CO2 incubator at 37 °C for 24 h to 48 h.
  4. KR activation (There are two ways for KR activation.)
    1. Local activation of one KR spot
      Local activation of one KR spot was performed with a 559 nm laser (1 mW/scan) of Olympus FV1000 confocal microscopy system in a selected area. One scan takes less than 1 sec. The final power (160 mJ) delivered to the KR (around 1 µm2) spot is around 6 mJ/µm2.
    2. Activation of KR in bulk cells
      Activation of KR in bulk cells was done by exposing cells to a 15 watt SYLVANIA cool white fluorescent bulb for 10 min in a stage UVP.
  5. The response of either GFP-tagged protein or endogenous protein of authors at the sites of tetR or TA-KR can be monitored in living cell.
  6. The response of endogenous protein at the sites of tetR or TA-KR can be monitored by immunostaining with the antibody.

Notes

For calculation of the dose that was delivered to the KillerRed spot:

  1. In the case of the 559 nm laser, the laser light was delivered to the selected area (around 25 m2) with 20 mJ/sec for 8 sec. Therefore, the final power (160 mJ) delivered to the KR (around 1 µm2) spot is around 6 mJ/µm2. For calculation of the dose that was delivered to the KillerRed spot based on the pixel size, the pixel size for irradiation is (0.138 µm/pixel) and the dwell time per pixel is (8 us/pixel). The irradiation is at 1.0 mW (1.0 mJ/s). With a dwell time of 8 us/pixel, this irradiates each pixel with 8.0 nJ/pixel/scan. Multiplying by the number of scans gives the total energy per pixel.
  2. In the case of fluorescent light activation, the rate of light is 15 J/m2/sec. With a 10 min light exposure, 9,000 J were delivered to the whole dish; the final power delivered to the KR (around 1 µm2) spot is around 9 mJ/µm2 upon light exposure. Cells were placed under a water bottle (height to light is 15 cm) to prevent an increase of temperature.

Acknowledgments

This protocol has been adapted from Lan et al. (2010) and Lan et al. (2014).

References

  1. Lan, L., Nakajima, S., Wei, L., Sun, L., Hsieh, C. L., Sobol, R. W., Bruchez, M., Van Houten, B., Yasui, A. and Levine, A. S. (2014). Novel method for site-specific induction of oxidative DNA damage reveals differences in recruitment of repair proteins to heterochromatin and euchromatin. Nucleic Acids Res 42(4): 2330-2345.
  2. Lan, L., Ui, A., Nakajima, S., Hatakeyama, K., Hoshi, M., Watanabe, R., Janicki, S. M., Ogiwara, H., Kohno, T. and Kanno, S.-i. (2010). The ACF1 complex is required for DNA double-strand break repair in human cells. Mol Cell 40(6): 976-987.
  3. Masui, O., Bonnet, I., Le Baccon, P., Brito, I., Pollex, T., Murphy, N., Hupe, P., Barillot, E., Belmont, A. S. and Heard, E. (2011). Live-cell chromosome dynamics and outcome of X chromosome pairing events during ES cell differentiation. Cell 145(3): 447-458.

简介

ROS诱导的DNA损伤在时间和空间背景下在活细胞中修复,并且染色质结构对于原位DNA修复过程的考虑是关键的。众所周知,染色质重塑因子参与许多DNA损伤修复途径,表明染色质重塑促进DNA损伤修复的重要性。到目前为止,还没有方法诱导活细胞中的位点特异性氧化性DNA损伤。因此,不知道DNA修复机制在活性或凝集的染色质中是否不同。我们最近建立了一种新的方法,DTG(损害靶向一个基因组位点),研究活性氧(ROS)诱导的DNA损伤活动细胞中的DNA损伤反应在一个基因组活性或无活性转录。为此,我们在U2OS细胞中在X染色体上整合了四环素响应元件(TRE)盒(〜90kb)(Lan等人,2010),然后融合KillerRed(KR)刺激的ROS诱导物,其可以特异性产生ROS诱导的DNA损伤,tet-阻遏物(tetR-KR,OFF)或转录激活物(TA-KR,ON)(Lan等人, ,2014)(图1)。 TetR-KR或TA-KR分别结合TRE盒并在异源或真核细胞状态下诱导ROS损伤。如何染色质状态调节DNA损伤反应过程可以通过使用这种强大的方法来检查。



图1. DTG系统的方案。 A.在U2OS TRE细胞系中tetR和TA标记的KR表达的方案。 为了在基因组中的特定基因座诱导ROS介导的损伤,我们将KR与四环素阻遏物融合以在U2OS细胞中的90kb四环素反应元件(TRE)阵列(共96个重复)中诱导ROS损伤。 B.T4R-KR在U2OS TRE细胞系中的表达。

材料和试剂

  1. U2OS-SCE细胞系(在我们的实验室制备)(Lan等人,2010)
    注意:
    1. 在该细胞系中,将200拷贝的pTRE/I-SceI整合到U2OS细胞中。
    2. 用Dulbecco's Modified Eagle's Medium High培养细胞 葡萄糖与稳定的L-谷氨酰胺(DMEM)(EuroClone S.p.A.P.IVA,目录 编号:ECM0103L)与10%胎牛血清(FBS)(Sigma-Aldrich, 目录号:F9665)。
    3. 对于细胞的制备,用不含Ca 2+和Mg 2+的PBS洗涤细胞。 2 + (EuroClone SpAPIVA,目录号:ECB4004L)进行胰蛋白酶消化,用胰蛋白酶/EDTA进行胰蛋白酶消化而不使用Ca (Thermo Fisher Scientific,目录号:BW17161E)。
  2. 质粒
    1. pBROAD3/tetR-KR
    2. pBROAD3/tetR-mCherry
    3. pBROAD3/TA-KR
    4. pBROAD3/TA-mCherry
      注意:pBROAD3/tetR-mcherry由Edith Heard博士提供(Masui et   et al。,2011); pBROAD3/tetR-KR,pBROAD3/TA-mCherry,pBROAD3/TA-KR 在我们的实验室(Lan等人,2014)。 基于序列   请求。
  3. GFP融合蛋白或内源性蛋白质
  4. Lipofectamine 2000(Life Technologies,目录号:12566-014)
  5. Opti-MEM(Life Technologies,目录号:51985-091)
  6. 35mm玻璃底培养皿(MatTek,目录号:P35GC-1.5-14-C)
  7. IMMOIL-F30CC(Chip Humphries,目录号:Z-81225)

设备

  1. 35毫米玻璃底培养皿
  2. 37℃,5%CO 2细胞培养箱中培养
  3. 具有488,555nm激光的Olympus FV1000共焦显微镜系统(OLYMPUS,型号:FV1000-FILTER DETECT; SYS.PACKAGE:IX81-1 405/M_AR/559/635N)
  4. PLAPON 60x油透镜(超级色差校正物/1.4NA FV)(OLYMPUS,目录号:FM1-U2B990)
  5. 热板(用于IX81/71/51/70/50的MATS-U52RA26,金属插件,HQ控制)(OLYMPUS,目录号:OTH-I0126)
  6. 15瓦冷白色荧光灯(OSRAM SYLVANIA)
  7. 阶段UVP(Upland,CA)

程序

  1. 将U2OS-SCE细胞以具有10%FBS的DMEM中的每个培养皿的5×10 5个近似密度接种到具有5%CO 2的37℃培养箱中的35mm玻璃底培养皿中,/sub>过夜。
  2. 对于GFP融合蛋白,使用Lipofectamine 2000与任何1μg的pBROAD3/tetR-KR,pBROAD3/tetR-mCherry,pBROAD3/TA-KR和pBROAD3/TA-mCherry共转染1μgGFP-融合蛋白。对于内源蛋白 ,只转染pBROAD3/tetR-KR,pBROAD3/tetR-mCherry,pBROAD3/TA-KR和pBROAD3/TA-mCherry中的任一种。 遵循Lipofectamine 2000的转染方案。
  3. 然后在37℃下在5%CO 2培养箱中孵育24小时至48小时。
  4. KR激活(KR激活有两种方式。)
    1. 本地激活一个KR点
      一个KR斑的局部激活 用559nm激光(1mW /扫描)的Olympus FV1000共聚焦进行 显微镜系统。 一次扫描需要不到1秒钟。 传送到KR(大约1μm2)的最终功率(160mJ)是 约6mJ /μm 2
    2. 批量电池中KR的激活
      激活 的KR通过将细胞暴露于15瓦的SYLVANIA来进行 冷白色荧光灯泡在阶段UVP 10分钟。
  5. 可以在活细胞中监测作者在tetR或TA-KR位点的GFP标记蛋白或内源蛋白的反应。
  6. 可以通过用抗体进行免疫染色来监测内源蛋白在tetR或TA-KR的位点处的反应。

笔记

用于计算输送到KillerRed斑点的剂量:

  1. 在559nm激光器的情况下,激光以20mJ/sec输送到选定区域(约25μm2/s)8秒。因此,传送到KR(大约1μm2)点的最终功率(160mJ)为大约6mJ /μm2/s。为了基于像素大小计算传送到KillerRed斑点的剂量,照射的像素大小为(0.138μm/像素),每个像素的驻留时间为(8μs/像素)。照射为1.0mW(1.0mJ/s)。在驻留时间为8us /像素的情况下,以8.0nJ /像素/扫描照射每个像素。乘以扫描次数得到每个像素的总能量。
  2. 在荧光激活的情况下,光速为15J/m 2/s/sec。在10分钟光照下,9,000J递送到整个培养皿;在曝光时传送到KR(大约1μm2)点的最终功率为大约9mJ /μm2。将细胞置于水瓶(高度为15cm)下以防止温度升高。

致谢

该协议已经由Lan等人(2010)和Lan等人(2014)修改。

参考文献

  1. Lan,L.,Nakajima,S.,Wei,L.,Sun,L.,Hsieh,CL,Sobol,RW,Bruchez,M.,Van Houten,B.,Yasui,A。和Levine,AS(2014) 。 用于位点特异性诱导氧化性DNA损伤的新方法揭示了修复蛋白募集到异染色质的差异, euchromatin。 Nucleic Acids Res。42(4):2330-2345。
  2. Lan,L.,Ui,A.,Nakajima,S.,Hatakeyama,K.,Hoshi,M.,Watanabe,R.,Janicki,SM,Ogiwara,H.,Kohno,T.and Kanno, 。 (2010)。 ACF1复合物是人类细胞中DNA双链断裂修复所必需的。 em> Mol Cell 40(6):976-987
  3. Masui,O.,Bonnet,I.,Le Baccon,P.,Brito,I.,Pollex,T.,Murphy,N.,Hupe,P.,Barillot,E.,Belmont,AS和Heard, 2011)。 活细胞染色体动力学和ES细胞分化期间X染色体配对事件的结果。 145(3):447-458
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wei, L., Nakajima, S., Levine, A. S. and Lan, L. (2014). Novel Method for Site-specific Induction of Oxidative DNA Damage to Study Recruitment of Repair Proteins to Heterochromatin and Euchromatin. Bio-protocol 4(11): e1140. DOI: 10.21769/BioProtoc.1140.
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