搜索

Nuclei Isolation from Nematode Ascaris
蛔虫线虫的细胞核分离   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Cell Reports
Aug 2016

Abstract

Preparing nuclei is necessary in a variety of experimental paradigms to study nuclear processes. In this protocol, we describe a method for rapid preparation of large number of relatively pure nuclei from Ascaris embryos or tissues that are ready to be used for further experiments such as chromatin isolation and ChIP-seq, nuclear RNA analyses, or preparation of nuclear extracts (Kang et al., 2016; Wang et al., 2016).

Keywords: Nuclei isolation (核分离), Embryos (胚胎), Tissue (组织), Nematodes (线虫), Ascaris (蛔虫)

Background

Nuclei isolation is often the first step in studying the molecular and biochemical aspects of nuclear events. Several methods have been developed to isolate nuclei from different tissues and cell types. However, few nuclei isolation protocols from nematodes other than C. elegans have been described (Ooi et al., 2010; Zanin et al., 2011; Haenni et al., 2012). Embryos of the parasitic nematode Ascaris have been used to prepare a variety of extracts for in vitro cell-free systems (Cohen et al., 2004; Lall et al., 2004). However, these extracts were typically whole cell extracts. Here we describe a method for preparation of nuclei from the nematode Ascaris.

Materials and Reagents

  1. 15 ml Falcon tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339651 )
  2. 50 ml Falcon tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339653 )
  3. 225 ml Falcon bottles (Corning, Falcon®, catalog number: 352075 )
  4. Ascaris
    Ascaris can be collected from slaughterhouses that process several thousand pigs a day and use the small intestines to make sausage casings. Ascaris can be picked up by hands (with gloves) from contents of intestine, which are pushed out to a tank by machines. Usually, it takes two persons 3-5 h to collect ~1,000 worms. Both fresh tissues and embryos can be obtained from these live worms. Female Ascaris can also be ordered from Carolina Biological, Living Zoology Department. These females are shipped dead on ice and the Ascaris zygotes can be obtained from the proximal uteri
  5. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: S318-1 )
  6. H2O (purified by Milli-Q® Integral Water Purification System)
  7. DAPI (Santa Cruz Biotechnology, catalog number: sc-3598 )
  8. Liquid nitrogen (Airgas, catalog number: NI NF160LT22 )
  9. Phenylmethanesulfonyl fluoride (PMSF) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 36978 )
  10. cOmpleteTM protease inhibitor cocktail (Roche Diagnostics, catalog number: 0493116001 )
  11. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-3 )
  12. Potassium chloride (KCl) (Fisher Scientific, catalog number: P217-500 )
  13. Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, catalog number: BP350-500 )
  14. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285-500 )
  15. Hydrochloric acid (HCl)
  16. 1 M Tris-HCl, pH 8.0 (Thermo Fisher Scientific, AmbionTM, catalog number: AM9856 )
  17. 1 M Tris-HCl, pH 7.0 (Thermo Fisher Scientific, AmbionTM, catalog number: AM9851 )
  18. Sodium hypochlorite solution (Fisher Scientific, catalog number: SS290-1 )
  19. Potassium hydroxide (KOH) (Fisher Scientific, catalog number: P250-1 )
  20. Magnesium chloride (MgCl2) (Fisher Scientific, catalog number: M33-500 )
  21. EGTA (Thermo Fisher Scientific, USB, catalog number: 15703 )
  22. Sucrose (Fisher Scientific, catalog number: BP220-212 )
  23. DTT (Fisher Scientific, catalog number: BP172-5 )
  24. Spermine (Sigma-Aldrich, catalog number: S3256 )
  25. Spermidine (Sigma-Aldrich, catalog number: S2626 )
  26. Nonidet P-40 (NP-40) (Alfa Aesar, Affymetrix/USB, catalog number: J19628 )
  27. Triton X-100 (Promega, catalog number: H5142 )
  28. Glycerol (Fisher Scientific, catalog number: BP229-1 )
  29. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250-10ML )
  30. EDTA (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17892 )
  31. PBS buffer (see Recipes)
  32. 1 M Tris-HCl, pH 7.8 (see Recipes)
  33. KOH/Bleach (0.4 N KOH/1.4% sodium hypochlorite) (see Recipes)
  34. Nuclei extraction buffer A (see Recipes)
  35. Nuclei extraction buffer B (see Recipes)
  36. Nuclei extraction buffer C (see Recipes)
  37. Nuclei storage buffer (see Recipes)

Equipment

  1. Erlenmeyer flask (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4112-0125 , 4112-0250 or 4112-0500 )
  2. 1 L beaker (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 12011000 )
  3. 4 L plastic beaker (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 12014000 )
  4. Shaking incubator (example, Lab-Line, model: 4629 )
  5. Fluorescence microscope (example, Nikon Instruments, model: TS100 )
  6. Refrigerated centrifuge (example, Thermo Fisher Scientific, model: SorvallTM LegendTM XTR )
  7. Metal dounce homogenizer (WHEATON, catalog number: 357574 )
  8. Mortar and pestle (example, Fisher Scientific, catalog numbers: S02591 and S02594 )

Procedure

  1. Isolation of embryos from Ascaris
    1. Ascaris embryos dissection
      1. Note that the anterior end of the female worms is thinner. Looking posterior from the anterior end, identify the genial girdle and cut female worms at or just posterior to the genital girdle (see Figure 1A). Males are typically smaller than females and have a strongly curved posterior (see Figure 1B).
      2. Gently pull the uteri completely out while holding on the posterior portion of the female until you feel tension. Examine the length of uterus, make cut and collect the proximal ¼ of the uterus into PBS (pH 7.0) in a ~1 L beaker on ice. The length of the proximal region will vary as the overall size of individual female worms can be quite variable (see Figure 1C).


        Figure 1. Ascaris embryo dissection. A. Female Ascaris. Light blue arrow indicates the genital girdle; Red arrow is the cutting area. B. Male Ascaris (left) vs. female Ascaris (right), male Ascaris are smaller and have a strong curve in the posterior (black arrow) (Modified from https://en.wikipedia.org/wiki/Ascaris_suum). C. Illustration of female worm cut at the genital girdle with the uteri pulled out. The proximal ¼ (white arrow) of the uterus is collected into PBS.

      3. Dispose of distal 3/4 of uterus.
      4. Rinse pooled uteri in a beaker three times with ice-cold PBS, pH 7.0 (hand over end to rinse; measure uterus volume in the beaker) (see Figure 2).


        Figure 2. Rinsed pooled Ascaris uteri

      5. Treat uteri with 0.5 N NaOH for ~1 h with gentle stirring at room temperature to keep the material suspended.
        The ratio of uteri/NaOH is ~50 ml packed uteri/0.6 L of 0.5 N NaOH. Stir to keep all material suspended.
      6. Allow ~45 min for solution to settle by gravity.
      7. Decant the solution slowly and carefully (as a safety precaution, decanted solution should be autoclaved before discarding). Note that there is often some white floating material present when you decant that you can pour out. The eggs stay down and when you see them nearing the spout stop decanting.
      8. Repeat the 0.5 N NaOH treatment and decant the supernatant.
      9. Transfer the egg suspension into 225 ml Falcon bottles. Collect the embryos at 1,250 x g for 10 min at 4 °C.
      10. Wash the embryos with ice-cold MilliQ water 3-4 times by spinning at 1,250 x g for 10 min at 4 °C.
      11. Wash with ice-cold PBS (pH 2.0) and collect the embryos at 1,250 x g for 10 min at 4 °C, finally resuspend and store the embryos in 5 volumes of ice-cold PBS (pH 2.0).
        Store at 4 °C (The embryos can be stored at 4 °C for at least 3-5 years) (see Figure 3).
        Note: Typical embryo yield for proximal uteri is ~20-25 ml/500 worms.


        Figure 3. Ascaris embryos. Keep Ascaris embryos in 5 volume of ice-cold PBS, pH 2.0. Store at 4 °C.

    2. Ascaris embryonation to prepare developmentally staged embryos for nuclei isolation
      1. Resuspend embryos and remove a volume of suspension appropriate for the experimental purpose (see Table 2 for the amount of nuclei to be used in different types of experiments).
        Note: Packed embryo volume will decrease by 60% after removal of the chitinous shell (Coat removal) and washing.
      2. Spin down embryos at 1,250 x g for 5 min at room temperature and discard supernatant.
      3. Wash embryos with 50 ml of PBS, pH 2.0, spin as above and discard supernatant.
      4. Resuspend embryos in ~20 volumes PBS, pH 2.0, and place them in sterile, disposable Erlenmeyer flask with cap on loose to facilitate aeration.
        Note: Volume of PBS should be no more than 30% of the flask. For example, the maximum volume of PBS should be 75 ml for 250 ml flask.
      5. Incubate embryos in an incubator at 30 °C shaking at 100 rpm for desired time of development (see timing of development Table 1).

        Table 1. Ascaris embryonation times at 30 °C with shaking
        Development time
        Predominant stage
        Other notes
        0 h
        1 cell
        Prior to pronuclear fusion
        24 h
        1 cell
        Pronuclei are fused
        46 h
        2 cell
        2-cell (82%)
        62-63 h
        2-4 cell
        2-cell (16%), 4-cell (49%), and 4-cell + (35%)
        96 h 
        10-26 cell 
        > 90%
        114 h
        32-64 cell 


      6. Examine embryos microscopically to ensure that the embryos have developed to the desired stages.
        Note: Embryos through the 8-cell stage of development can be easily staged via light microscopy, whereas later stages typically require DAPI nuclear staining via fluorescence microscopy to define the cell number and developmental stage (Wang et al., 2014).
    3. Chitinous shell (coat) removal of Ascaris embryos
      1. Pellet embryos (from step A2) in 15 or 50 ml Falcon tubes at 1,250 x g for 5 min at room temperature. Remove supernatant.
      2. Resuspend embryos in 15-20x pellet volumes or 50 ml of freshly made 0.4 N KOH/1.4% sodium hypochlorite (solution should be pre-warmed to 30 °C).
      3. Transfer suspension back to original Erlenmeyer flask and incubate for 90 min at 30 °C in incubator shaker with shaking at 100 rpm.
      4. Pellet embryos in 15 or 50 ml Falcon tubes at 1,250 x g for 5 min at room temperature.
      5. Wash embryos 5 times with 15-20x pellet volumes of ice-cold PBS, pH 7.0 by gentle resuspension and centrifugation as in step A1d.
      6. Estimate packed embryo volume from pelleted embryos. The volume of final embryos after decoating is typically about 60% of the starting volume (see Figures 4A and 4B).


        Figure 4. Ascaris embryos. A. One cell stage Ascaris embryo. There is thick chitinous shell (coat) outside the embryo membrane. B. One cell stage Ascaris embryo after coat removal. Chitinous shell is removed and embryo membrane is still there. C. Ascaris embryos after homogenate.

  2. Preparation of Ascaris tissue for nuclei isolation
    1. Ascaris dissection
      Ascaris can be collected from a pig slaughterhouse and placed in 30 °C PBS (pH 7.0) while being transported to the lab. It is important to keep the worms at 30 °C to maintain viability.
      Note: Ascaris can be kept alive in 30 °C PBS, pH 7.0 for at least 2-3 days. However, it is better to dissect the worms on the same day to get fresh tissues.
      1. Rinse worms with warm PBS, pH 7.0.
      2. Male Ascaris can be dissected to obtain spermatids, intestine, testis, and the remaining tissue (the carcass, which includes muscle, hypodermis, pharynx, and neurons). Female Ascaris can be dissected to obtain ovary/oviduct, oocytes, uterus, intestine, and carcass. Tissues should be frozen in liquid nitrogen (without PBS), and stored at -80 °C. Note that the reproductive systems can be up to a meter long and different regions can be identified and subdivided for analysis.

  3. Nuclei isolation
    1. Nuclei isolation from Ascaris embryos.
      1. Suspend ~900 μl of decoated embryos in 10 ml of nuclei extraction buffer A and centrifuge at 1,200 x g for 5 min at 4 °C. Note that ~10 million embryos can be obtained from ~1 ml packed decoated embryos (~20 mature female Ascaris). The number of nuclei will depend on the developmental stage of the embryos. (See Table 2 for the amount of nuclei necessary for different types of experiments.)

        Table 2. Protein, RNA, and DNA content as a function of Ascaris nuclei and amounts needed for ChIP experiments
        Experiments
        Nuclear protein (10 μg)
        Nuclear RNA (10 μg)
        DNA (10 ng)
        ChIP/ChIP-seq
        Nuclei
        ~105
        ~105
        ~104
        ~106

      2. Remove supernatant. Resuspend the 900 μl cell pellet in 10 ml of ice-cold nuclei extraction buffer B with 0.5 mM PMSF and cOmpleteTM protease inhibitor cocktail. Transfer the suspension into a 15 ml WHEATON metal dounce homogenizer kept on ice.
      3. Homogenize with 10 passes.
        Note: Examine embryos under a microscope to make sure over 80% are broken. If not, homogenize further (see Figure 4C).
      4. Transfer the suspension to 15 ml Falcon tubes and spin at 750 x g for 10 min at 4 °C in Sorvall Legend XT with swinging bucket rotor. Remove supernatant.
        Notes:
        1. Comparison of the number of nuclei in the supernatant to pellet (equivalent volumes) should show very high nuclei enrichment in pellets (some nuclei remain in supernatant).
        2. If you want to collect a cytoplasmic extract, retain the supernatant. For cytoplasmic extracts, we typically clarify the supernatant at 21,000 x g at 4 °C for 15 min, transfer aliquouts to pre-cooled microfuge tubes, freeze the tubes in liquid nitrogen, and store at -80 °C.
      5. Resuspend nuclear pellets in 10 ml of ice-cold nuclei extraction buffer A, centrifuge at 750 x g for 10 min at 4 °C in Sorvall Legend XT with swinging bucket rotor. Repeat twice and after the 3rd centrifugation step remove as much supernatant as possible from the nuclear pellet.
      6. Resuspend pellets in 10 ml of ice cold nuclei extraction buffer C, centrifuge at 2,000 x g for 15 min at 4 °C in Sorvall Legend XT in swinging bucket rotor. Remove supernatant and examine supernatant for nuclei. The majority of nuclei should be in pellet.
      7. The isolated nuclei can be used immediately or frozen in storage buffer in liquid nitrogen and stored at -80 °C for future experiments.
        Note: See isolation of Ascaris embryos, embryonation, and preparation of embryos for nuclei isolation in Procedure C.
    2. Nuclei isolation from Ascaris tissue
      1. Grind Ascaris tissue from 3-5 worms (~105-106 nuclei, depends on differences in the tissues) to a fine powder in liquid nitrogen using mortar and pestle. (See Table 2 for amount of nuclei should be used for different purpose of experiments.)
      2. Wash Ascaris tissue powder in 10 ml of ice-cold PBS and centrifuge at 1,200 x g for 5 min at 4 °C.
      3. Repeat step C2b twice.
      4. Isolate nuclei as described above in Procedure A.
        Note: The volume of buffer at each step should be optimized based on volume of tissue powder, use the same amount of buffer as used for the decoated embryos.

Notes

  1. Dissection of 1,500 worms takes ~4-5 h for 2 people. For efficient dissection and preventing bacterial growth, isolate embryos immediately upon worms and keep them cold.
  2. Ascaris is known to cause infection in human; hence proper precautionary measure should be followed to protect yourself and other lab members from infection. It has been reported that the pig Ascaris, Ascaris suum, can infect humans leading to pulmonary infection. Isolation of eggs from female Ascaris is not a direct infection threat. The infective stages of Ascaris are the L2/L3 stages that develop inside eggs. Ascaris eggs isolated from the uteri of females require 3-4 weeks development at 30 °C with appropriate aeration to become infective. Thus, if the embryos are not embryonated to infective larvae, they do not impose an infection risk. Although unlikely and very rare, it remains possible that some egg contamination could occur during their isolation from the uteri of females. Therefore, safety protocols to protect laboratory workers including personal protection devices, dedicated work places, sterilization of material coming in contact with Ascaris, and treatment of benches and material to remove Ascaris eggs to prevent their development to infective stages as followed. Finally, exposure to adult worms may lead to anaphylactic shock. Lab personnel should be informed of all the risks of working with Ascaris including symptoms of anaphylactic shock and its treatment. In addition, as a precaution people should work in pairs with live adult worms to ensure that another person is present in the event that someone develops anaphylactic shock.
  3. Depending on the tissues and size of the nuclei this protocol can be modified and optimized to work with other nematodes.
  4. The large size of the Ascaris allows different parts of the worm tissues to be isolated and used for nuclei extraction. However, for most other small nematodes, the whole worm is used for nuclei isolation.

Recipes

  1. PBS buffer
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Add ddH2O up to 1 L
    Adjust the pH to 7.0 or 2.0 with HCl
  2. 1 M Tris-HCl, pH 7.8
    15 ml 1 M Tris-HCl, pH 7.0
    16 ml 1 M Tris-HCl, pH 8.0
  3. KOH/Bleach (0.4 N KOH/1.4% sodium hypochlorite)
    35.2 ml of sodium hypochlorite solution
    2.24 g KOH
    Add ddH2O up to 100 ml
  4. Nuclei extraction buffer A
    1.5 mM MgCl2
    1 mM EGTA
    0.5 M sucrose
    10 mM KCl
    20 mM Tris-HCl, pH 7.8
    1 mM DTT
    1 mM spermine
    1 mM spermidine
    Add ddH2O to desired final volume
  5. Nuclei extraction buffer B
    Add the list to nuclei extraction buffer A
    0.15% NP-40
    0.1% Triton X-100
    Add ddH2O to desired final volume
    Add 0.5 mM PMSF and 1x cOmpleteTM protease inhibitor cocktail immediately prior use
  6. Nuclei extraction buffer C
    1.5 mM MgCl2
    1 mM EGTA
    1 M sucrose
    10 mM KCl
    20 mM Tris-HCl, pH 7.8
    1 mM DTT
    1 mM spermine
    1 mM spermidine
    Add dd H2O to desired final volume
  7. Nuclei storage buffer
    40% glycerol
    50mM Tris-HCl
    5mM MgCl2
    0.05% 2-mercaptoethanol
    1 mM DTT
    1 mM EDTA
    Add ddH2O to desired final volume

Acknowledgments

This work was supported in part by NIH grants to R.E.D. (AI0149558 and AI114054).

References

  1. Cohen, L. S., Mikhli, C., Friedman, C., Jankowska-Anyszka, M., Stepinski, J., Darzynkiewicz, E. and Davis, R. E. (2004). Nematode m7GpppG and m32,2,7GpppG decapping: activities in Ascaris embryos and characterization of C. elegans scavenger DcpS. RNA 10(10): 1609-1624.
  2. Haenni, S., Ji, Z., Hoque, M., Rust, N., Sharpe, H., Eberhard, R., Browne, C., Hengartner, M. O., Mellor, J., Tian, B. and Furger, A. (2012). Analysis of C. elegans intestinal gene expression and polyadenylation by fluorescence-activated nuclei sorting and 3'-end-seq. Nucleic Acids Res 40(13): 6304-6318.
  3. Kang, Y., Wang, J., Neff, A., Kratzer, S., Kimura, H. and Davis, R. E. (2016). Differential chromosomal localization of centromeric histone CENP-A contributes to nematode programmed DNA elimination. Cell Rep 16(9): 2308-2316.
  4. Lall, S., Friedman, C. C., Jankowska-Anyszka, M., Stepinski, J., Darzynkiewicz, E. and Davis, R. E. (2004). Contribution of trans-splicing, 5' -leader length, cap-poly(A) synergism, and initiation factors to nematode translation in an Ascaris suum embryo cell-free system. J Biol Chem 279(44): 45573-45585.
  5. Ooi, S. L., Henikoff, J. G. and Henikoff, S. (2010). A native chromatin purification system for epigenomic profiling in Caenorhabditis elegans. Nucleic Acids Res 38(4): e26.
  6. Wang, J., Garrey, J. and Davis, R. E. (2014). Transcription in pronuclei and one- to four-cell embryos drives early development in a nematode. Curr Biol 24(2): 124-133.
  7. Zanin, E., Dumont, J., Gassmann, R., Cheeseman, I., Maddox, P., Bahmanyar, S., Carvalho, A., Niessen, S., Yates, J. R., 3rd, Oegema, K. and Desai, A. (2011). Affinity purification of protein complexes in C. elegans. Methods Cell Biol 106: 289-322.

简介

在各种实验范例中准备核是必要的,以研究核过程。在本协议中,我们描述了一种从准备用于进一步实验(如染色质分离和ChIP-seq,核RNA)的蛔虫胚胎或组织快速制备大量相对纯的核的方法分析或准备核提取物(Kang等人,2016; Wang等人,2016)。

背景 核分离通常是研究核事件的分子和生物化学方面的第一步。已经开发了几种方法来分离来自不同组织和细胞类型的细胞核。然而,除了C以外的线虫的细胞核分离方案很少。已经描述了线索,(Ooi等人,2010; Zanin等人,2011; Haenni等人,2012, )。已经使用寄生线虫蛔虫的胚胎来制备用于体外无细胞系统的各种提取物(Cohen等人,2004) ; Lall等人,2004)。然而,这些提取物通常是全细胞提取物。在这里,我们描述了从线虫蛔虫制备细胞核的方法。

关键字:核分离, 胚胎, 组织, 线虫, 蛔虫

材料和试剂

  1. 将15ml Falcon管(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:339651)
  2. 50ml Falcon管(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:339653)
  3. 225ml Falcon瓶(Corning,Falcon ®,目录号:352075)
  4. 蛔虫
    蛔虫可以从每天处理数千只猪的屠宰场收集,并使用小肠制成香肠肠衣。蛔虫可以从肠内收集的手(用手套)取出,并用机器将其推出。通常需要两个人3-5小时收集〜1000个蠕虫。新鲜的组织和胚胎都可以从这些活蠕虫获得。女性蛔虫也可以从Carolina Biological,Living Zoology Department订购。这些女性在冰上死亡,蛔虫合子可以从近端子宫获得
  5. 氢氧化钠(NaOH)(Fisher Scientific,目录号:S318-1)
  6. H 2 O(通过Milli-Q 积分水净化系统纯化)
  7. DAPI(Santa Cruz Biotechnology,目录号:sc-3598)
  8. 液氮(Airgas,目录号:NI NF160LT22)
  9. 苯基甲烷磺酰氟(PMSF)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:36978)
  10. cOmplete TM蛋白酶抑制剂混合物(Roche Diagnostics,目录号:0493116001)
  11. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-3)
  12. 氯化钾(KCl)(Fisher Scientific,目录号:P217-500)
  13. 磷酸二氢钠(Na 2 HPO 4)(Fisher Scientific,目录号:BP350-500)
  14. 磷酸二氢钾(KH 2 PO 4)(Fisher Scientific,目录号:P285-500)
  15. 盐酸(HCl)
  16. 1M Tris-HCl,pH8.0(Thermo Fisher Scientific,Ambion TM,目录号:AM9856)
  17. 1M Tris-HCl,pH7.0(Thermo Fisher Scientific,Ambion TM,目录号:AM9851)
  18. 次氯酸钠溶液(Fisher Scientific,目录号:SS290-1)
  19. 氢氧化钾(KOH)(Fisher Scientific,目录号:P250-1)
  20. 氯化镁(MgCl 2)(Fisher Scientific,目录号:M33-500)
  21. EGTA(Thermo Fisher Scientific,USB,目录号:15703)
  22. 蔗糖(Fisher Scientific,目录号:BP220-212)
  23. DTT(Fisher Scientific,目录号:BP172-5)
  24. 精胺(Sigma-Aldrich,目录号:S3256)
  25. 亚精胺(Sigma-Aldrich,目录号:S2626)
  26. Nonidet P-40(NP-40)(Alfa Aesar,Affymetrix/USB,目录号:J19628)
  27. Triton X-100(Promega,目录号:H5142)
  28. 甘油(Fisher Scientific,目录号:BP229-1)
  29. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250-10ML)
  30. EDTA(Thermo Fisher Scientific,Thermo Scientific TM,目录号:17892)
  31. PBS缓冲液(参见食谱)
  32. 1M Tris-HCl,pH 7.8(参见食谱)
  33. KOH /漂白剂(0.4N KOH/1.4%次氯酸钠)(参见食谱)
  34. 核提取缓冲液A(参见食谱)
  35. 核提取缓冲液B(参见食谱)
  36. 核提取缓冲液C(参见食谱)
  37. 核存储缓冲区(见配方)

设备

  1. 锥形瓶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:4112-0125,41112-0250或4112-0500)
  2. 1L烧杯(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:12011000)
  3. 4L塑料烧杯(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:12014000)
  4. 摇动培养箱(例如,Lab-Line,型号:4629)
  5. 荧光显微镜(例如,Nikon Instruments,型号:TS100)
  6. 冷冻离心机(例如,Thermo Fisher Scientific,型号:Sorvall TM Legend TM XTR)
  7. 金属均质器(WHEATON,目录号:357574)
  8. 砂浆和杵(例如,Fisher Scientific,目录号:S02591和S02594)

程序

  1. 从蛔虫分离胚胎
    1. 蛔虫胚胎解剖
      1. 注意,雌虫的前端较薄。从前端向后看,识别脐带,并在生殖器腰带之前或之后切割雌性蠕虫(见图1A)。男性通常小于女性,并且具有强烈弯曲的后方(见图1B)。
      2. 轻轻地将子宫完全拉出,同时握住女性的后部,直到感到紧张。检查子宫的长度,在冰上约1L的烧杯中切片并将子宫的近端¼收集到PBS(pH7.0)中。近端区域的长度将变化,因为单个雌性蠕虫的总体尺寸可能非常不同(见图1C)。


        图1.蛔虫胚胎解剖。 A.女性蛔虫。浅蓝色箭头表示生殖器腰带;红色箭头是切割区域。 (男)蛔虫(左)与女性蛔虫(右),男性蛔虫较小,后部有强烈的曲线箭头)(修改自 https://en.wikipedia.org/wiki/Ascaris_suum )。 C.女性蜗牛的切割在生殖器腰带与子宫拔出。将子宫的近端¼(白色箭头)收集到PBS中
      3. 处理远端3/4的子宫。
      4. 用冰冷的PBS,pH7.0(接头冲洗;测量烧杯中的子宫体积)在烧杯中冲洗合并的子宫3次(见图2)。


        图2. Rinsed pooled 蛔虫 uteri

      5. 用0.5N NaOH处理子宫〜1小时,在室温下轻轻搅拌,以保持材料悬浮 子宫/NaOH的比例是约50ml 50ml/10升0.5N NaOH。搅拌以保持所有物料停产。
      6. 允许约45分钟的溶液通过重力沉降。
      7. 缓慢小心地倾倒溶液(作为安全预防措施,倾倒溶液应在弃置之前进行高压灭菌)。请注意,当您倾倒时,您可以倾吐出来,通常会出现一些白色的浮动物质。鸡蛋留下来,当你看到他们靠近喷口停止滗水时。
      8. 重复0.5N NaOH处理并倒出上清液。
      9. 将蛋悬浮液转移到225ml Falcon瓶中。在4℃下以1,250×g/g收集胚胎10分钟。
      10. 用冰冷的MilliQ水洗涤胚胎3-4次,通过在4℃下以1,250×g旋转10分钟。
      11. 用冰冷的PBS(pH2.0)洗涤,并在4℃下以1,250×g收集胚胎10分钟,最后将胚胎重新悬浮并储存在5体积的冰冷PBS(pH2.0)中, 。
        储存于4°C(胚胎可以在4°C保存至少3-5年)(见图3)。
        注意:近端子宫的典型胚胎产量为〜20-25毫升/500个蠕虫。


        图3.蛔虫胚胎蛔虫胚胎放入5体积冰冷的PBS,pH2.0中。在4°C储存。

    2. 蛔虫胚胎制备用于细胞核分离的发育阶段胚胎
      1. 重悬胚胎并移除适合实验目的的一定体积的悬浮液(参见表2,用于不同类型实验的细胞核数量)。
        注意:去除几丁质壳(去除衣壳)和洗涤后,包装的胚胎体积将减少60%。
      2. 在室温下将胚胎转移到1,250×g 5分钟,弃去上清液。
      3. 用50ml PBS洗涤胚胎,pH 2.0,如上旋转并弃去上清液。
      4. 将胚胎重悬于约20体积的PBS,pH为2.0,并将其置于无菌的一次性锥形瓶中,盖上松动以便于通气。
        注意:PBS的体积不应超过烧瓶的30%。例如,对于250ml烧瓶,PBS的最大体积应为75ml。
      5. 在培养箱中孵育30℃的胚胎,以100rpm摇动所需的发育时间(参见开发时间表1)。

        表1.蛔虫在30℃下振荡时的胚胎时间
        开发时间
        主要阶段
        其他注释
        0 h
        1个单元格
        原核融合之前
        24小时
        1个单元格
        原核融合
        46小时
        2个单元格
        2单元格(82%)
        62-63小时
        2-4细胞
        2细胞(16%),4细胞(49%)和4细胞+(35%)
        96 h 
        10-26 cell 
        > 90%
        114小时
        32-64单元格 


      6. 检查胚胎在显微镜下确保胚胎发育到所需阶段 注意:通过8个细胞发育阶段的胚胎可以通过光学显微镜进行轻松分期,而后期阶段通常需要通过荧光显微镜进行DAPI核染色来定义细胞数量和发育阶段(Wang等,2014)。
    3. 胚胎壳(外套)去除蛔虫胚胎
      1. 在室温下,在1550或50ml Falcon管中以1,250×g的颗粒胚(来自步骤A2)5分钟。去除上清液
      2. 以15-20倍的丸粒体积重新悬浮胚胎或50ml新鲜制备的0.4N KOH/1.4%次氯酸钠(溶液应预热至30℃)。
      3. 将悬浮液转移回原来的三角烧瓶中,并在30℃下在培养箱摇床中以100rpm摇动孵育90分钟。
      4. 在1550或50毫升Falcon管中以1,250×g的颗粒胚在室温下5分钟。
      5. 用15-20倍小粒体积的冰冷的PBS,pH7.0,通过如步骤A1d轻轻重新悬浮和离心,洗涤胚胎5次。
      6. 从胚胎胚胎估计包装胚胎体积。脱漆后最终胚胎的体积通常为起始体积的约60%(见图4A和4B)。


        图4.蛔虫胚胎 A.一个细胞阶段蛔虫胚胎。在胚胎膜外面有厚厚的壳多糖壳(外套)。 B.除虫后的一个细胞阶段蛔虫胚胎。去除壳多糖壳,仍然存在胚胎膜。 C.蛔虫匀浆后的胚胎。

  2. 准备用于细胞核分离的蛔虫组织
    1. 蛔虫解剖
      蛔虫可以从猪屠宰场收集并置于30℃PBS(pH7.0)中,同时运送到实验室。将蠕虫保持在30°C以维持生命力是很重要的。
      注意:蛔虫可以在30℃PBS,pH 7.0中保存至少2-3天。然而,最好在同一天解剖蠕虫以获得新鲜的组织。
      1. 用温热的PBS,pH 7.0冲洗蠕虫。
      2. 可以解剖睾丸,以获得精子细胞,肠,睾丸和剩余的组织(包括肌肉,皮下组织,咽和神经元的胴体)。女性蛔虫可以解剖以获得卵巢/输卵管,卵母细胞,子宫,肠和胴体。组织应在液氮(不含PBS)中冷冻,并储存于-80°C。请注意,生殖系统长达一米,可以识别和细分不同的区域进行分析。

  3. 核分离
    1. 从蛔虫胚胎进行核分离。
      1. 将约900μl脱蛋白的胚悬浮于10ml细胞核提取缓冲液A中,并在4℃下以1,200×g离心5分钟。请注意,可以从〜1毫升包装的脱色胚胎(〜20成熟女性蛔虫)获得约1000万个胚胎。核的数量将取决于胚胎的发育阶段。 (不同类型实验所需的核数量见表2)
        表2.蛋白质,RNA和DNA含量作为蛔虫细胞核和ChIP实验所需的量
        实验
        核蛋白(10μg)
        核RNA(10μg)
        DNA(10 ng)
        ChIP/ChIP-seq
        核 -
        〜10 5
        〜10 5
        〜10 4
        〜10 6

      2. 去除上清液。将900μl细胞沉淀重悬于含有0.5 mM PMSF和cOm完全蛋白酶抑制剂混合物的10 ml冰冷核提取缓冲液B中。将悬浮液转移到保持在冰上的15ml WHEATON金属均质器中。
      3. 均匀10次。
        注意:在显微镜下检查胚胎,以确保超过80%被破坏。如果没有,进一步均质(见图4C)。
      4. 将悬浮液转移到15ml Falcon管中,并在Sorvall Legend XT中用旋转叶轮转子在4℃下以750×g旋转10分钟。去除上清液 注意:
        1. 上清液中的细胞核数量(等效体积)的比较应该显示细胞核中的非常高的核富集(一些细胞核保留在上清液中)。
        2. 如果要收集细胞质提取物,请保留上清液。对于细胞质提取物,我们通常以21,000 x g在4℃澄清上清液15分钟,将分批转移到预冷却的微量离心管中,将管冷冻在液氮中,并储存在-80℃。
      5. 将核颗粒重悬在10ml冰冷的核提取缓冲液A中,在具有摆动的桶式转子的Sorvall Legend XT中,在4℃下以750×g离心10分钟。重复两次,然后3小时离心步骤从核沉淀中除去尽可能多的上清液。
      6. 将沉淀物重悬于10ml冰冷核提取缓冲液C中,在旋转桶转子的Sorvall Legend XT中以4℃在2,000×g下离心15分钟。去除上清液并检查核上清液。大多数核应该是颗粒。
      7. 分离的核可以立即使用或在液氮中储存缓冲液中冷冻,并储存在-80°C进行未来实验。
        注意:参见步骤C中分离蛔虫胚胎,胚胎分离和用于细胞核分离的胚胎的制备。
    2. 从蛔虫组织中的核分离
      1. 来自3-5个蠕虫(〜10 5 -10 6 细胞核的蛔虫组织取决于组织的差异)与细粉中的细粉使用砂浆和杵的液氮。 (见表2中核的量应用于不同的实验目的。)
      2. 在10毫升冰冷的PBS中洗涤蛔虫组织粉末,并在4℃下以1,200×g离心5分钟。
      3. 重复步骤C2b两次。
      4. 隔离核,如上述程序A所述 注意:每个步骤的缓冲液体积应根据组织粉末的体积进行优化,使用与去皮胚胎相同量的缓冲液。

笔记

  1. 解决1500个蠕虫需要4-5个小时的2人。为了有效的解剖和预防细菌生长,请在蠕虫后立即分离胚胎,并保持冷。
  2. 已知蛔虫在人体中引起感染;因此应遵循适当的预防措施,以保护您和其他实验室成员免受感染。据报道,蛔虫蛔虫蛔虫可以感染导致肺部感染的人。从女性蛔虫中分离鸡蛋不是直接的感染威胁。蛔虫的感染阶段是卵内发育的L2/L3阶段。从女性子宫中分离的蛔虫需要在30℃下开发3-4周才能适当曝气以成为感染性的。因此,如果胚胎没有被胚胎传染给感染性幼虫,那么它们不会产生感染风险。虽然不太可能和非常罕见,但仍然有可能在与女性子宫隔离期间发生一些鸡蛋污染。因此,保护实验室工作人员的安全协议,包括个人保护装置,专用工作场所,与蛔虫接触的材料的灭菌,以及治疗长凳和材料以去除蛔虫鸡蛋以防止其发展成为感染阶段。最后,暴露于成虫可能导致过敏性休克。应向实验室人员通报所有与蛔虫有关的风险,包括过敏性休克及其治疗的症状。此外,作为预防措施,人们应与活的成年蠕虫成对配对,以确保另一人在有人发生过敏性休克的情况下存在。
  3. 根据细胞核的组织和大小,该协议可以修改和优化,以与其他线虫一起使用
  4. 大规模的蛔虫允许不同部分的蠕虫组织被分离并用于核提取。然而,对于大多数其他小线虫,整个蠕虫用于核分离。

食谱

  1. PBS缓冲液
    8克NaCl
    0.2克KCl
    1.44g Na 2 HPO 4
    0.24g KH 2 PO 4
    添加ddH 2 O至1 L
    用HCl将pH调至7.0或2.0
  2. 1M Tris-HCl,pH7.8。
    15ml 1M Tris-HCl,pH7.0
    16ml 1M Tris-HCl,pH8.0
  3. KOH /漂白剂(0.4N KOH/1.4%次氯酸钠)
    35.2ml次氯酸钠溶液
    2.24克KOH
    将ddH 2 O加至100 ml
  4. 核提取缓冲液A
    1.5mM MgCl 2
    1 mM EGTA
    0.5 M蔗糖
    10 mM KCl
    20mM Tris-HCl,pH7.7
    1 mM DTT
    1mM精胺
    1mM亚精胺
    将ddH 2 O添加到所需的最终卷
  5. 核提取缓冲液B
    将列表添加到核提取缓冲区A
    0.15%NP-40
    0.1%Triton X-100
    将ddH 2 O添加到所需的最终卷
    在使用之前先添加0.5 mM PMSF和1x cOmplete™蛋白酶抑制剂混合物
  6. 核提取缓冲液C
    1.5mM MgCl 2
    1 mM EGTA
    1 M蔗糖
    10 mM KCl
    20mM Tris-HCl,pH7.7
    1 mM DTT
    1mM精胺
    1mM亚精胺
    将dd H <2> O加入到所需的最终体积
  7. 核存储缓冲区
    40%甘油
    50mM Tris-HCl
    5mM MgCl 2
    0.05%2-巯基乙醇
    1 mM DTT
    1 mM EDTA
    将ddH 2 O添加到所需的最终卷

致谢

这项工作得到NIH授予R.E.D.的部分支持。 (AI0149558和AI114054)。

参考

  1. Cohen,LS,Mikhli,C.,Friedman,C.,Jankowska-Anyszka,M.,Stepinski,J.,Darzynkiewicz,E.and Davis,RE(2004)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15383679"target ="_ blank">线虫m 7 GpppG和m <3> 2,2,7 GpppG脱蛋白:蛔虫的活动胚胎和表征。 elegans 清道夫DcpS。 10(10):1609-1624。
  2. Haenni,S.,Ji,Z.,Hoque,M.,Rust,N.,Sharpe,H.,Eberhard,R.,Browne,C.,Hengartner,MO,Mellor,J.,Tian,B.and Furger ,A.(2012)。  分析 C。线虫通过荧光激活的核分选和3'-末端序列进行肠基因表达和多聚腺苷酸化。核酸Res 40(13):6304-6318。 >
  3. Kang,Y.,Wang,J.,Neff,A.,Kratzer,S.,Kimura,H.and Davis,RE(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27545882"target ="_ blank">着丝粒组蛋白的差异染色体定位CENP-A有助于线虫编程的DNA消除。细胞Rep 16 (9):2308-2316。
  4. Lall,S.,Friedman,CC,Jankowska-Anyszka,M.,Stepinski,J.,Darzynkiewicz,E.and Davis,RE(2004)。< a class ="ke-insertfile"href ="http: /www.ncbi.nlm.nih.gov/pubmed/15322127"target ="_ blank">转录,5'长度,cap-poly(A)协同作用的贡献和线虫翻译的起始因子, em>蛔虫 suum胚胎细胞系统。 J Biol Chem 279(44):45573-45585。
  5. Ooi,SL,Henikoff,JG和Henikoff,S。(2010)。一种用于秀丽隐杆线虫表观遗传学分析的天然染色质纯化系统。核酸Res 38(4):e26。
  6. Wang,J.,Garrey,J.and Davis,RE(2014)。  原核和一至四细胞胚胎的转录驱动线虫的早期发育。 24(2):124-133。
  7. Zanin,E.,Dumont,J.,Gassmann,R.,Cheeseman,I.,Maddox,P.,Bahmanyar,S.,Carvalho,A.,Niessen,S.,Yates,JR,3rd,Oegema,和Desai,A.(2011)。亲和纯化蛋白质复合物。 elegans 。方法细胞周期 106:289-322。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kang, Y., Wang, J. and Davis, R. E. (2017). Nuclei Isolation from Nematode Ascaris. Bio-protocol 7(9): e2262. DOI: 10.21769/BioProtoc.2262.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。