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DNA Damage Sensitivity Assays in Caenorhabditis elegans
秀丽隐杆线虫的DNA损伤敏感性检测   

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Abstract

C. elegans has served as a genetically tractable multicellular model system to examine DNA damage-induced genotoxic stress which threatens genome integrity. Importantly, the high degree of conservation shared between worms and humans offers the advantage that findings about DNA damage-induced cell cycle arrest/checkpoint response and DNA double-strand break repair in worms are applicable to human studies. Here, we describe simple DNA damage sensitivity assays to quantify the response of C. elegans to diverse types of DNA damaging agents. These assays have provided important insights into the mechanisms of function for factors such as ZTF-8 that are involved in DNA damage repair and response in the C. elegans germline. These DNA damage sensitivity assays rely on the straightforward readouts of either egg or larval lethality and involve the use of various DNA damaging agents. We use γ-irradiation (γ-IR), which produces DNA double-strand breaks (DSBs), camptothecin (CPT), which induces single-strand breaks, nitrogen mustard (HN2), which produces interstrand crosslinks (ICLs), hydroxyurea (HU), which results in replication fork arrest thus preventing DNA synthesis, and UV-C, which causes photoproducts (pyrimidine dimers). See Table 1. Comparisons between the relative sensitivity/resistance observed in, for example, mutants compared to wild type, for various DNA damaging agents allows for inferences regarding potential repair pathways being affected.

Keywords: DNA damage(DNA损伤), C. elegans(线虫), DNA repair(DNA修复), Genotoxin(遗传毒物), Germline(生殖系)

Materials and Reagents

  1. E.coli OP50 (Carolina, catalog number 155073 )
  2. M9 (He, 2011)
  3. NGM agar media (He, 2011)
  4. M9 500 ml + Triton X-100 50 μl
  5. Mechlorethamine hydrochloride (Sigma-Aldrich, catalog number 122564 )
  6. Camptothecin (Sigma-Aldrich, catalog number C9911 )
  7. Hydroxyurea (Sigma-Aldrich, catalog number H8627 )
  8. Triton X-100 (Sigma-Aldrich, catalog number T8787 )
  9. 20% Alkaline Hypochlorite Solution (see Recipes)

Equipment

  1. 24-well plates (OLYMPUS, catalog number 25-102 )
  2. Short tip disposable glass Pasteur pipets (VWR International, catalog number 14673-010 )
  3. Petri dishes (60 x 15 mm) (VWR International, catalog number 25384-092 )
  4. Sealing film (Parafilm M, catalog number: PM996 )
  5. Nutator mixer (Clay Adams, model: 1105 Mixer )
  6. 20 °C incubator (Thermo Fisher Scientific, Precision 815)
  7. 37 °C shaking incubator (New Brunswick, Innova 4330 )
  8. 37 °C incubator ((VWR Internationa, 1510E)
  9. Benchtop centrifuge for spinning 15 ml tubes (Lanmet, Hermle Z, model: 400K )
  10. Stereo microscope (Leica Microsystems, model: MZ75 )
  11. UV crosslinker (Stratagene, model: 2400 Stratalinker) with 254 nm UV bulbs
  12. γ-IR irradiator (Shepherd & Associates, Mark 1 Cs137 irradiator)

Procedure

  1. Preparation of genotoxins
    DNA damaging agents are generally toxic and require extra safety precautions when working with them. Wear double gloves and a lab coat. Always work in a laminar flow and label and seal your containers in hard-walled robust containers. Make fresh solutions to avoid possible degradation and always seal the tubes, even for short-term storage. Caution must be exercised to assure that all waste materials are disposed of properly.
    In this protocol, solid/agar media is used for UVC, γ-IR, and HU sensitivity assays, while CPT and HN2 treatments are performed in liquid culture (Table 1). Defects in meiotic pachytene are addressed by analysis 26-28 h post exposure (Jaramillo-Lambert et al., 2010). Mitotic repair failures are examined by analysis 48-72 h post exposure.
    1. Pyrimidine dimmers: UV irradiation treatment can be performed utilizing the 2400 Stratalinker. Worms are exposed to 0 to 150 J/m2 of UVC. The lids are removed from the Petri dishes to ensure better UV penetration. Most UV crosslinkers have a UV sensor inside so caution must be taken to avoid blocking/disrupting the UV sensor by placing plates next to it. After treatment, worm plates are wrapped in foil to avoid potential photo recovery. Known sensitive strains: slx-1 (tm2644) (Saito et al., 2012) and him-18 (tm2181) (Saito et al., 2012).
    2. DNA double-strand breaks: To assess for γ-IR sensitivity, doses ranging between 0 to 150 Gy are generally used. Find the radiation dose rate (Gy/h) for your irradiator to calculate a time for exposure. Known sensitive strains: him-18 (tm2181) (Saito et al., 2012), brc-1 (tm1145) (Ward et al., 2007), and ztf-8 (tm2176) (Kim and Colaiacovo, 2014).
    3. Replication arrest: Hydroxyurea is soluble in water. Dissolve HU completely with a good amount of water (~10 ml) and mix it into autoclaved NGM agar media (1 L) when it has cooled down to 55-50 °C. HU is hygroscopic and must be sealed and stored in a desiccator. HU sensitivity is assessed by placing animals on NGM plates seeded with OP50 (E.coli) and containing 0 to 40 mM HU for 20 h. Known sensitive strains: clk-2 (mn159) (Bailly et al., 2010),and ztf-8 (tm2176) (Kim and Colaiacovo, 2014).
    4. Single-strand breaks: Camptothecin is not soluble in water and instead is soluble in DMSO at 10 mg/ml. Higher concentrations require heat for it to completely enter into solution (10 min at 95 °C). It is advisable to prepare a lower concentration to avoid heating a toxic material. The final range of concentrations used for camptothecin is 0 to 1,000 nM in M9 buffer containing OP50. M9 solution at a pH of 6.0 was reported to deliver higher sensitivity compared with pH 7.0 by way of better impeding topoisomerase activity (Kessler and Yanowitz, 2014). Known sensitive strains: him-18 (tm2181) (Saito et al., 2012) and brc-1 (tm1145) (Ward et al., 2007).
    5. Interstrand cross-links: Nitrogen mustard (HN2) is soluble in water. For nitrogen mustard (HN2) sensitivity, animals are treated with 0 to 150 μM of HN2 in M9 buffer containing OP50. Known sensitive strains: brc-1 (tm1145) (Ward et al., 2007) and slx-1 (tm2644) (Saito et al., 2012).

      Table 1. Known DNA damage sensitive strains for genotoxins. A summary of genotoxins, the lesions they produce, and sensitive strains. The media used for this protocol is also indicated.


  2. Worm preparation
    C. elegans strains are cultured in NGM plates (60 x 15 mm petri dishes) seeded with OP50 E. coli at 20 °C under standard conditions as described in Brenner (1974). In brief, 10 ml NGM agar containing plates are prepared and dried in storage bins kept at room temperature for 4-5 days before being seeded with OP50 E. coli and placed overnight in a 37 °C incubator. The N2 Bristol strain is used as the wild-type control together with sensitive mutants as described in Table 1.

  3. Daily procedures
    Basic procedures for both liquid and solid exposures are similar, as illustrated in Figure 1, although the liquid exposures require additional washes before and after mutagen treatment. Calculate the total number of worms required for an assay. For example, for three doses of exposure, approximately 75 worms of each genotype are required (5 worms/plate x 5 plates x 3 doses). However, since some worms can be lost as they are prone to attaching to plastics, such as pipette tips and Eppendorf tubes, and given the potential lethality that may occur due to exposure to the genotoxins, it is recommended to have 2 times more worms in general.

    Figure 1. A schematic view of daily procedures for DNA damage sensitivity assay. Synchronized young adults are prepared by either egg prep by bleaching worms at day 1 or hand picking L4 worms at day 4. As illustrated, basic procedures for both liquid and solid exposures are similar. Note that liquid exposure requires additional washes before and after exposure to mutagens. See section B. Daily procedures for details.

    Day 1
    1. To prepare synchronized L1 stage larvae, gravid adult hermaphrodites are washed off from the plates by adding 1-2 ml of M9/plate. Leave the plates with M9 for 30-60 sec to dislodge the worms. Alternatively, for a small-scale test, L4 worms can be picked with a platinum wire instead of washing off the plate. However, synchronizing via bleaching removes potential fungal contamination of the worm which can be problematic when counting worms. The total number of adult worms required might vary, but 40 adults are required to obtain ~160 L1 larvae. If you have synchronized L4 worms ready for the assays, skip this step and start from step C10.
    2. Use a glass Pasteur pipette to transfer worms to 15 ml falcon tubes. (*Short tip length Pasteur pipettes are easier to manipulate. It is convenient to mark the 300 μl line on the 15 ml tubes used in step C3.)
    3. Spin the worms in a swing-bucket centrifuge (30-60 sec at ~1,000 rpm or ~210 rcf) and remove the supernatant by gently aspirating the M9. Leave ~ 300 μl of solution at the bottom to avoid disturbing worm pellets.
    4. Add 3 ml of 20% alkaline hypochlorite solution and gently swirl the tube. Most worms will break open and release their eggs in ~ 5 min. Do not bleach too long to avoid killing the eggs.
    5. Add ~15 ml of M9 to each tube and invert the tubes a few times.
    6. Repeat step C3 three more times to remove the alkaline hypochlorite solution.
    7. Add M9 up to 15 ml to the tubes and incubate overnight at 20 °C with gentle rocking (Nutator mixer). Seal the cap with Parafilm to avoid leaking.
    Day 2
    1. Repeat step C3.
    2. From the ~300 μl of solution remaining at the bottom following centrifugation and removal of the supernatant, distribute proper amount of liquid containing L1 worms to an OP50 seeded plate. Count the number of worms and make sure not to have either too many or not enough worms in a plate. Aim for ~50 worms for each seeded plate. If you start from 40 gravid adults, distribute 50 μl to 6 seeded plates (total 300 μl) after gently mixing the pellet by vortexing. Incubate ~48 h at 20 °C until worm reaching the L4 larval stage.
    Day 3
    Briefly monitor worm growth. Make sure worms are not starved.
    Day 4
    1. Worms will be at the L4 stage. Incubate an additional 18-20 h and young adult worms will be ready for further analysis. In general, pick twice the number of worms for each dose per genotype. For example, pick 150 worms of N2 worms for three doses (25 worms x 3 doses x 2 times).
    2. For exposure in liquid media (CPT and HN2): Inoculate OP50 in 20 ml LB media in a 50 ml conical tube and incubate in a shaker incubator overnight at 37 °C. Note that a 20 ml culture is enough for 3 genotypes (two controls + sample) with 3 doses of exposures in liquid.
    Day 5
    1. For exposure in solid media (UV, γ-IR and HU): Age-matched young adults are transferred to OP50 seeded plates for γ-IR and UV exposures. Age-matched young adults are transferred to HU-containing seeded plate for HU sensitivity assay (Figure 2A).
      For γ-IR and UV treatments, worms are exposed to the DNA damaging source and then incubated for 18-20 h at 20 °C. Go to step C19.
      For HU treatment, worms are incubated in HU containing media for 18-20 h at 20 °C. Go to step C19. Avoid a thick bacterial lawn as it might affect UV/IR penetration. Only use plates seeded with overnight grown bacteria. See Notes.


      Figure 2. A schematic representation of DNA damage sensitivity assay design with solid (A) and liquid (B) exposure. A. For UVC, γ-IR and HU treatments, animals are exposed on seeded solid NGM media. B. One set of 9 wells is used for CPT (Green) or HN2 (Red) sensitivity. Animals are exposed to genotoxins suspended in liquid culture. Dose of genotoxin is indicated by the gradient color. Empty circles indicate empty wells. Positive (known sensitive mutants, see Table1) and negative controls (wild type N2) as well as mutant worms are incubated in the indicated column. Short curved lines represent animals in panels A and B. Squares represent bacterial lawns in panel A.

    2. For exposure in liquid media (CPT and HN2): Spin down 20 ml of OP50 (OD600 >2.0) in LB media and remove supernatant. Add 4 ml of M9 and vortex to resuspend the pellet. For example, split 1.3 ml bacterial solution to 3 Eppendorf tubes for 3 doses of exposure including one for a mock exposure.
    3. Add genotoxin to the tubes containing OP50 for a 1.25x higher than the desired final concentration and Vortex briefly. For example, for final 100 μM, make 125 μM 1.3 ml HN2 + OP50.
    4. Dispensing genotoxins: Transfer 400 μl to each of 3 wells of a 24-well plate (horizontal direction). Repeat this step for each dose. See Figure 2B for an example of 3 doses x 3 genotypes x 2 genotoxins.
    5.  Dispensing worms: Transfer age-matched young adults to 15 ml tubes and wash with 15 ml M9 + Triton X-100 two times followed by two washes with only M9.
      Aspirate M9 gently and leave ~ 300 μl solution at the bottom to avoid disturbing worm pellets. Transfer 100 μl worm to each of 3 vertical wells from step C15 (100 μl x 3 wells, see Figure 2B). This will result in a total of 500 μl/well with the desired final concentration of genotoxin plus animals (400 + 100 μl). Repeat this step for each genotype.
    6. Wrap the plate with foil and incubate at 20 °C for 18-20 h with gentle shaking.
    Day 6
    1. Repeat steps C2-3 twice.
    2. Transfer worms to seeded NGM plates. (Gently swirl the plates to spread any excess liquid so it can be more efficiently absorbed by the media. Alternatively, aspirate the liquid carefully without sucking up worms.) Allow for recovery by incubating for 3 h at 20 °C.
    3. Pick worms and split 5 per plate and incubate at 20 °C for ~4 h (5 plates/dose/genotype). Avoid a thick bacterial lawn as it makes the counting harder. See Notes.
    4. P0 worms can be removed and discarded or can be used for further analysis such as immunostaining or DAPI staining. Count the number of eggs laid (for wild type N2, 70-100 eggs are expected per plate). Draw a grid with a dark colored marker on a Petri dish lid, which you can place under your plate to facilitate scoring. See Figure 3.


      Figure 3. Schematic representation of how to score numbers of eggs laid and hatched on NGM agar plates. Using a dark colored marker draw a grid on a Petri dish lid. This lid can be placed under your plate to facilitate scoring under a stereomicroscope.

    Day 7
    1. After incubation at 20 °C for 20-24 h, count numbers of unhatched/dead eggs and hatched F1 worms. For counting larvae, it is easier to count when they become L2/L3, however unhatched eggs will be degraded and therefore more difficult to see and count if you wait longer.

      If necessary, continue to count larvae and adults at Day 9. Represent the data as either embryonic viability or larval lethality as shown in Figure 4.

      Figure 4. DNA damage sensitivity assay results for ztf-8 mutants. Representative data using graphs adapted from Kim and Colaiacovo (2014). Relative embryonic viability and larval lethality for ztf-8 and clk-2 mutants compared to wild type after treatment with the indicated doses of γ-IR and HU. The results from two independent experiments (biological replicates) are shown. Asterisks indicate statistical significance calculated by two-tailed Mann-Whitney test, 95% confidence interval. Error bars represent standard error of the mean.

Notes

  1. Instead of spinning down worms using a centrifuge, worms can also be harvested by sedimentation at the bench for ~5 min.
  2. In general, over 25 animals were plated, 5 per plate. Each DNA damage-inducing condition was replicated at least twice in independent experiments.
  3. To avoid a thick bacterial lawn, seed 50 μl of overnight grown OP50 (OD600>2.0) liquid culture to NGM plates and spread it with a glass rod. Incubate 12-14 h at 37 °C.

Recipes

  1. 20% alkaline hypochlorite solution (45 ml)
    Make fresh each time
    24.75 ml ddH2O
    11.25 ml 1 M NaOH
    9.0 ml Bleach (non germicidal)

Acknowledgments

We thank Doris Lui, Elisabeth Altendorfer and Jinmin Gao for proof reading the manuscript. This work was supported by National Institutes of Health grants R01GM072551 and R01GM105853 to MPC. This protocol has been adapted from our previous work (Kim and Colaiacovo, 2014).

References

  1. Bailly, A. P., Freeman, A., Hall, J., Declais, A. C., Alpi, A., Lilley, D. M., Ahmed, S. and Gartner, A. (2010). The Caenorhabditis elegans homolog of Gen1/Yen1 resolvases links DNA damage signaling to DNA double-strand break repair. PLoS Genet 6(7): e1001025.
  2. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77(1): 71-94.
  3. He, F. (2011). Common worm media and buffers. Bio-protocol Bio101: e55.
  4. Jaramillo-Lambert, A., Harigaya, Y., Vitt, J., Villeneuve, A. and Engebrecht, J. (2010). Meiotic errors activate checkpoints that improve gamete quality without triggering apoptosis in male germ cells. Curr Biol 20(23): 2078-2089.
  5. Kessler, Z. and Yanowitz, J. (2014). Methodological considerations for mutagen exposure in C. elegans. Methods 68(3): 441-449.
  6. Kim, H. M. and Colaiacovo, M. P. (2014). ZTF-8 interacts with the 9-1-1 complex and is required for DNA damage response and double-strand break repair in the C. elegans germline. PLoS Genet 10(10): e1004723.
  7. Saito, T. T., Mohideen, F., Meyer, K., Harper, J. W. and Colaiacovo, M. P. (2012). SLX-1 is required for maintaining genomic integrity and promoting meiotic noncrossovers in the Caenorhabditis elegans germline. PLoS Genet 8(8): e1002888.
  8. Ward, J. D., Barber, L. J., Petalcorin, M. I., Yanowitz, J. and Boulton, S. J. (2007). Replication blocking lesions present a unique substrate for homologous recombination. EMBO J 26(14): 3384-3396.

简介

C。 elegans 作为一种遗传易损的多细胞模型系统,以检查DNA损伤诱导的基因毒性应激,这威胁着基因组的完整性。重要的是,蠕虫和人类之间共享的高程度的保护提供了关于DNA损伤诱导的细胞周期停滞/检查点反应和DNA双链断裂修复蠕虫中的发现适用于人类研究的优势。在这里,我们描述简单的DNA损伤灵敏度测定以量化C的反应。 elegans 到各种类型的DNA损伤剂。这些测定提供了对涉及DNA损伤修复和在C中的应答的因子如ZTF-8的功能的机制的重要见解。 elegans 种系。这些DNA损伤敏感性测定依赖于卵或幼虫致死性的直接读出,并涉及使用各种DNA损伤剂。我们使用产生DNA双链断裂(DSB)的γ-照射(γ-IR),诱导单链断裂的喜树碱(CPT),氮芥(HN <2>),其产生链间交联(ICL),羟基脲(HU),其导致复制叉停止,从而防止DNA合成,和UV-C,其引起光产物(嘧啶二聚体)。参见表1.在例如突变体与野生型相比,在各种DNA损伤剂中观察到的相对灵敏度/抗性之间的比较允许关于潜在修复途径受影响的推论。

关键字:DNA损伤, 线虫, DNA修复, 遗传毒物, 生殖系

材料和试剂

  1. 大肠杆菌 OP50(Carolina,目录号155073)
  2. M9(He,2011)
  3. NGM琼脂培养基(He,2011)
  4. M9 500ml + Triton X-10050μl
  5. 盐酸二乙胺(Sigma-Aldrich,目录号122564)
  6. 喜树碱(Sigma-Aldrich,目录号C9911)
  7. 羟基脲(Sigma-Aldrich,目录号H8627)
  8. Triton X-100(Sigma-Aldrich,目录号T8787)
  9. 20%碱性次氯酸盐溶液(参见配方)

设备

  1. 24孔板(OLYMPUS,目录号25-102)
  2. 短端一次性玻璃巴斯德移液管(VWR International,目录号14673-010)
  3. 培养皿(60×15mm)(VWR International,目录号25384-092)
  4. 密封膜(Parafilm M,目录号:PM996)
  5. Nutator mixer(Clay Adams,型号:1105 Mixer)
  6. 20℃培养箱(Thermo Fisher Scientific,Precision 815)
  7. 37℃振荡培养箱(New Brunswick,Innova 4330)
  8. 37℃培养箱((VWR Internationa,1510E))
  9. 用于旋转15ml管(Lanmet,Hermle Z,型号:400K)的台式离心机
  10. 立体显微镜(Leica Microsystems,型号:MZ75)
  11. UV交联剂(Stratagene,型号:2400 Stratalinker),用254nm UV灯泡
  12. γ-IR照射器(Shepherd& Associates,Mark 1 Cs 137 照射器)

程序

  1. 基因毒素的制备
    DNA损伤剂通常是有毒的,在使用它们时需要额外的安全预防措施。穿双手套和实验室外套。始终在层流中工作,并在硬壁坚固的容器中标记和密封容器。制备新鲜的溶液以避免可能的降解,并始终密封管,即使短期储存。必须小心谨慎,以确保所有的废料都得到妥善处理 在该方案中,固体/琼脂培养基用于UVC,γ-IR和HU灵敏度测定,而CPT和HN 2处理在液体培养中进行(表1)。减数分裂长期的缺陷通过暴露后26-28小时的分析来解决(Jaramillo-Lambert等人,2010)。有丝分裂修复失败通过暴露后48-72小时的分析进行检查
    1. 嘧啶二聚体:可以利用紫外线照射处理 2400 Stratalinker。将蠕虫暴露于0至150J/m 2的UVC。的 从培养皿中取出盖子以确保更好的UV渗透。 大多数紫外线交联剂内部有一个紫外线传感器,所以必须小心  通过将板放置在其旁边来避免阻挡/中断UV传感器。 处理后,蜗形板被包裹在箔中以避免电位 照片恢复。已知的敏感菌株: slx-1 ( tm2644 )(Saito 2012)和 him-18 ( tm2181 )(Saito等人,2012)。
    2. DNA双链 断裂:评估γ-IR灵敏度,剂量范围在0至150之间 Gy通常使用。查找您的辐射剂量率(Gy/h) 以计算曝光时间。已知的敏感菌株: him-18 ( tm2181 )(Saito ,2012),
      ( tm1145 )(Ward 等 2007)和 ztf-8 ( tm2176 )(金和Colaiacovo,2014)。
    3. 复制  抑制:羟基脲溶于水。完全溶解HU 良好量的水(〜10ml),并将其混合到高压灭菌的NGM琼脂培养基中 (1L),当其已经冷却至55-50℃时。 HU是吸湿性的,必须是  密封并储存在干燥器中。 HU灵敏度评估 将动物置于用OP50(大肠杆菌)接种的NGM平板上,  至40mM HU 20小时。已知的敏感菌株: clk-2 ( mn159 )(Bailly et  al。,2010)和 ztf-8 ( tm217 6)(金和Colaiacovo,
    4. 单链断裂:喜树碱不溶于水,而是 以10mg/ml溶于DMSO。较高的浓度需要热量 它完全进入溶液(95℃10分钟)。这是可取的 以制备较低的浓度,避免加热有毒物质。的 喜树碱的最终浓度范围为0至1,000nM in M9缓冲液中含有OP50。 M9溶液,pH6.0 通过更好的方式提供比pH 7.0更高的灵敏度 阻碍拓扑异构酶活性(Kessler和Yanowitz,2014)。已知 敏感菌株: him-18 ( tm2181 )(Saito等人,2012)和 brc-1 tm1145 )(Ward等人,,2007)。
    5. 链间交联:氮 芥子(HN 2)可溶于水。对于氮芥(HN 2) 在M9缓冲液中用0至150μM的HN 2 S处理动物 含OP50。已知的敏感菌株: brc-1 ( tm1145 )(Ward等人 2007)和 slx-1 ( tm2644 )(Saito ,2012)。
      表1.已知 DNA损伤敏感菌株的基因毒素。基因毒素, 它们产生的损伤,以及敏感菌株。用于此的媒体  还指示协议。


  2. 蠕虫准备
    C。 elegans 菌株在接种OP50 E的NGM平板(60×15mm培养皿)中培养。如在Brenner(1974)中所述的标准条件下在20℃温育大肠杆菌。简言之,制备10ml含NGM琼脂的板,并在用OP50E接种之前在保持在室温的储存箱中干燥4-5天。并在37℃培养箱中放置过夜。 N2 Bristol菌株与表1所述的敏感突变体一起用作野生型对照
  3. 日常程序
    液体和固体暴露的基本程序是相似的,如图1所示,尽管液体暴露需要在诱变处理之前和之后的额外的洗涤。计算测定所需的蠕虫总数。例如,对于三个剂量的暴露,需要每个基因型约75虫(5虫/板×5板×3剂)。然而,由于一些蠕虫可能会丢失,因为它们倾向于附着到塑料,如移液管尖端和Eppendorf管,并给出潜在的致死率,可能会由于暴露于基因毒素,可能会发生,建议有2倍的蠕虫一般。

    图1. DNA损伤灵敏度测定的日常程序的示意图。 通过在第1天漂白蠕虫的蛋制备或在第4天手动挑选L4蠕虫来准备同步的年轻成年人。如图所示, 液体和固体暴露的基本程序是相似的。注意,液体暴露需要在暴露于诱变剂之前和之后的额外的洗涤。详情请参阅B.日常程序
    第1天
    1. 为了准备同步的L1阶段幼虫,妊娠成体雌雄同体  通过加入1-2ml的M9 /板从板中洗去。离开 板与M9 30-60秒以驱逐蠕虫。或者,对于a  小规模试验,L4蠕虫可以用铂丝代替 的洗掉板。但是,通过漂白同步会删除 潜在的真菌污染的蠕虫,这可能是有问题的时候  计数蠕虫。所需的成虫总数可能不同, 但40名成年人需要获得〜160 L1幼虫。如果你有 同步L4蠕虫准备好测定,跳过此步骤并开始 从步骤C10。
    2. 使用玻璃巴斯德吸管转移蠕虫 15 ml falcon管。 (*短端长度的巴斯德移液器更容易 操作。 在15ml管上标记300μl管是方便的 在步骤C3中使用。)
    3. 在秋千桶离心机中旋转蠕虫 (在〜1,000rpm或〜210rcf下30-60秒),并除去上清液 轻轻吸出M9。 留下〜300微升的溶液在底部 避免扰动蠕虫丸。
    4. 加入3ml 20%碱性 次氯酸盐溶液并轻轻旋转试管。 大多数蠕虫会破裂 打开和释放他们的鸡蛋在〜5分钟。 不要漂白太长时间避免 杀死鸡蛋。
    5. 每管加入〜15 ml M9,倒置几次
    6. 重复步骤C3三次以除去碱性次氯酸盐溶液。
    7. 向管中加入最多15ml的M9,并在20℃下孵育过夜   温和摇动(Nutator mixer)。 用石蜡膜密封帽避免 泄漏。
    第2天
    1. 重复步骤C3。
    2. 从〜300μl的溶液保留在 离心并除去上清液后, 将适量的含有L1蠕虫的液体分配到OP50种子   盘子。 计数蠕虫的数量,并确保不要有 许多或没有足够的蠕虫在盘子里。 瞄准每个种子〜50蠕虫 盘子。 如果你从40个妊娠成年人开始,分配50微升到6个种子 通过涡旋轻轻混合沉淀后,将板(总共300μl)。 在20°C孵育〜48 h,直到蠕虫到达L4幼虫阶段。
    第3天
    简要监测蠕虫生长。 确保蠕虫不饿。
    第4天
    1. 蠕虫将处于L4阶段。 额外孵化18-20小时和年轻 成虫会准备进一步分析。 一般来说,选择两次 每个基因型的每个剂量的蠕虫数。 例如,选择150 N2蠕虫的蠕虫三剂(25虫×3剂×2次)
    2. 对于在液体介质(CPT和HN 2)中的暴露:接种在20mL的OP50中 LB培养基在50ml锥形管中,并在摇床培养箱中孵育 在37℃过夜。 注意,20ml培养物足以用于3种基因型 (两个对照+样品)与3个剂量的液体中的暴露。
    第5天
    1. 用于在固体介质(UV,γ-IR和HU)中暴露:年龄匹配的年轻成人 转移到OP50接种板用于γ-IR和UV暴露。 将年龄匹配的年轻成年人转移到含HU的接种板中 用于HU灵敏度测定(图2A) 对于γ-IR和UV处理, 蠕虫暴露于DNA损伤来源,然后孵育 在20℃下18-20小时。 转到步骤C19。
      对于HU治疗,蠕虫是 在含有HU的培养基中在20℃温育18-20小时。 转到步骤C19。 避免厚的细菌草坪,因为它可能会影响UV/IR渗透。 只要 使用接种过夜生长的细菌的平板。 见注释。


      图 2.DNA损伤灵敏度测定设计的示意图 与固体(A)和液体(B)暴露。 A.对于UVC,γ-IR和HU 处理,将动物暴露于接种的固体NGM培养基上。 B.一套  9个孔用于CPT(绿色)或HN 2(红色)灵敏度。动物是 暴露于悬浮在液体培养基中的基因毒素。基因毒素的剂量是 由渐变颜色指示。空心圆圈表示空孔。 阳性(已知敏感突变体,参见表1)和阴性对照 (野生型N2)以及突变体蠕虫在所示的温育 柱。短曲线代表图A和B中的动物 表示面板A中的细菌草坪
    2. 暴露于 液体培养基(CPT和HN 2):将20ml的OP50(OD <600> 2.0)旋转到 LB培养基并除去上清液。加入4毫升M9,涡旋重悬 颗粒。例如,将1.3ml细菌溶液分流到3 Eppendorf 管,用于3次剂量的暴露,包括一次用于模拟暴露。
    3. 向含有OP50的管中加入基因毒素,高于1.25倍 所需的最终浓度和涡旋。 例如,for 最后100μM,制备125μM1.3ml HN 2 + OP50
    4. 点胶 基因毒素:将400μl转移至24孔板的3个孔中的每个孔中 (水平方向)。 对每个剂量重复此步骤。 参见图2B 例如3剂量×3基因型×2基因毒素
    5.  分配蠕虫:将年龄相符的年轻成年人转移到15毫升管和 用15ml M9 + Triton X-100洗涤两次,然后用两次洗涤 只有M9。
      轻轻吸出M9,离开〜300μl溶液 底部以避免扰动蠕虫丸。 转移100微升蠕虫到每个   来自步骤C15的3个垂直孔(100μl×3个孔,参见图2B)。 这个 将导致总共500μl/孔与期望的最终 基因毒素加动物(400 +100μl)的浓度。 重复此步骤   为每个基因型
    6. 用箔包裹板,并在20℃下温和振荡孵育18-20小时。
    第6天
    1. 重复步骤C2-3两次。
    2. 转移蠕虫到种子NGM板。 (轻轻旋转板,以扩散任何多余的液体,所以它可以更多 有效地被介质吸收。 或者,抽吸液体 仔细,不吸吮蠕虫。)允许通过孵化恢复 在20℃下保持3小时
    3. 挑取蠕虫,每板5分,孵化 在20℃下〜4小时(5个平板/剂量/基因型)。 避免厚的细菌草坪   因为它使得计数更难。 见注释。
    4. P <0> 蠕虫可以 移除并丢弃或可用于进一步分析,例如 免疫染色或DAPI染色。 计数鸡蛋的数量(野生   类型N2,预期每板70-100个鸡蛋)。 用黑色绘制网格 在培养皿盖子的色的标志,您能安置在您的板材下   以方便记分。 见图3.


      图3.原理图 表示如何对NGM上铺设和孵化的蛋的数量进行评分 琼脂平板上。使用深色标记在培养皿上画一个网格 盖。 这个盖子可以放在你的板下,以方便刻下 立体显微镜
    第7天
    1. 在20°C孵育20-24小时后,计数未孵化/死亡 鸡蛋和孵化的F1蠕虫。对于计数幼虫,它更容易计数 当它们变为L2/L3时,未孵化的卵将被降解 因此如果你等待的时间更长,更难看到和计数
      如果 必要的,在第9天继续计数幼虫和成虫 数据如胚胎生存力或幼虫致死率所示 图4.

      图4. ztf-8突变体的DNA损伤敏感性测定结果。代表性数据使用从Kim和 Colaiacovo(2014)。在用野生型处理后,与野生型相比,ztf-8和em-clk-2突变体的相对胚胎生存力和幼虫致死率 指示剂量的γ-1R和HU。 结果从两个独立 实验(生物学重复)。 星号表示 通过双尾Mann-Whitney检验计算的统计学显着性,95%   置信区间。 误差线表示平均值的标准误差。

笔记

  1. 代替使用离心机旋转蠕虫,蠕虫也可以通过在工作台上沉降约5分钟来收获。
  2. 一般来说,接种超过25只动物,每板5只。 每个DNA损伤诱导条件在独立实验中至少复制两次
  3. 为了避免厚的细菌菌苔,将50μl过夜生长的OP50(OD 600至2.0)液体培养物种至NGM平板,并用玻璃棒涂布。 在37℃孵育12-14小时。

食谱

  1. 20%碱性次氯酸盐溶液(45ml) 每次清新
    24.75ml ddH 2 O
    11.25ml 1M NaOH
    9.0 ml漂白剂(无杀菌)

致谢

我们感谢Doris Lui,Elisabeth Altendorfer和Jinmin Gao为阅读手稿的证明。这项工作是由国家卫生研究院拨款R01GM072551和R01GM105853支持MPC。这个协议已经从我们以前的工作(Kim和Colaiacovo,2014)改编。

参考文献

  1. Bailly,A.P.,Freeman,A.,Hall,J.,Declais,A.C.,Alpi,A.,Lilley,D.M.,Ahmed,S.and Gartner,A。(2010)。 Gen1/Yen1裂解酶的 Caenorhabditis elegans同系物将DNA损伤信号传导DNA双链断裂修复。 PLoS Genet 6(7):e1001025。
  2. Brenner,S。(1974)。 Caenorhabditis elegans的遗传学 遗传学 77(1):71-94。
  3. 他,F.(2011)。 常见的蠕虫介质和缓冲液 生物协议 Bio101:e55。
  4. Jaramillo-Lambert,A.,Harigaya,Y.,Vitt,J.,Villeneuve,A。和Engebrecht,J。(2010)。 邪恶错误激活了改善配子的检查点 质量而不触发雄性生殖细胞中的凋亡。 Curr Biol 20(23):2078-2089。
  5. Kessler,Z.和Yanowitz,J。(2014)。 在C中诱变剂暴露的方法学考虑因素。 elegans 。 方法 68(3):441-449。
  6. Kim,H.M。和Colaiacovo,M.P。(2014)。 ZTF-8与9-1-1复合物相互作用,是DNA损伤反应和双重在C中的断裂修复。 elegans germline。 PLoS Genet 10(10):e1004723。
  7. Saito,T.T.,Mohideen,F.,Meyer,K.,Harper,J.W.and Colaiacovo,M.P.(2012)。 SLX-1是维持基因组完整性和在秀丽隐杆线虫中促进减数分裂非交换所必需的 germline。 PLoS Genet 8(8):e1002888。
  8. Ward,J.D.,Barber,L.J.,Petalcorin,M.I.,Yanowitz,J。和Boulton,S.J。(2007)。 复制性阻断病变为同源重组提供了独特的底物。 EMBO J 26(14):3384-3396。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kim, H. and Colaiácovo, M. P. (2015). DNA Damage Sensitivity Assays in Caenorhabditis elegans. Bio-protocol 5(11): e1487. DOI: 10.21769/BioProtoc.1487.
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