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Heavy Metal Stress Assay of Caenorhabditis elegans
秀丽隐杆线虫重金属胁迫检测   

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Abstract

Organisms have developed many protective systems to reduce the toxicity from heavy metals. The nematode Caenorhabditis elegans has been widely used to determine the protective mechanisms against heavy metals. Responses against heavy metals can be monitored by expression of reporter genes, while sensitivity can be determined by quantifying growth or survival rate following exposure to heavy metals.

Keywords: Caenorhabditis elegans(秀丽隐杆线虫), Arsenic(砷), Cadmium(镉), Copper(铜)

Background

Some heavy metals, such as arsenic, cadmium and mercury, are known to be harmful to the majority of organisms including humans (Valko et al., 2005). To reduce the toxicity by these metals, the organisms have developed various protective systems. The nematode Caenorhabditis elegans has been used to understand the mechanisms of protection against heavy metals. Previous studies have revealed that many genes, such as detoxification enzymes, transcription factors and signaling factors, are involved in the protection from heavy metals in this organism (Broeks et al., 1996; Mizuno et al., 2004; Inoue et al., 2005; Schwartz et al., 2010). Determinations of viability and growth, in addition to measurements of reporter gene expression, are usually used to monitor the effects of heavy metals in C. elegans. In this protocol, we describe the methods for assays for arsenic, copper and cadmium using C. elegans.

Materials and Reagents

  1. Latex glove (KCWW, Kimberly-Clark, catalog number: 57330 )
  2. Petri dishes 60 x 15 mm (Iwaki, catalog number: 1010-060 )
  3. 1.5 ml plastic tubes (Eppendorf, catalog number: 3810X )
  4. 1 ml pipetman tips (Thermo Fisher Scientific, Thermo Scientific, catalog number: 111-N-Q )
  5. 0.2 ml pipetman tips (Thermo Fisher Scientific, Thermo Scientific, catalog number: 110-N-Q )
  6. Slide glass (Matsunami Glass, catalog number: S2227 )
  7. Paper tape
  8. Cover glass (Matsunami Glass, 18 x 18, Thickness No.1)
  9. Corning 50 ml centrifuge tubes (Corning, catalog number: 4558 )
  10. Petri dishes 35 x 15 mm (Iwaki, catalog number: 1000-035 )
  11. Pasteur pipet, 5 inch (Iwaki, catalog number: IK-PAS-5P )
  12. Parafilm (Wako Pure Chemical Industries, catalog number: 535-02443 )
  13. Autoclave tape
  14. pH probe (As One, catalog number: 2-347-05 )
  15. 10 ml glass tubes (Iwaki, catalog number: 09183982 )
  16. 99.98% Platinum Wire, ø0.2 mm (Nilaco, catalog number: 351325 )
  17. OP50 E. coli bacteria (University of Minnesota, C. elegans Genetics Center, N/A)
  18. Worm strains (can be obtained from CGC: see Table 1 for example)
  19. Calcium chloride dihydrate (CaCl2·2H2O) (Wako Pure Chemical Industries, catalog number: 031-00435 )
  20. Potassium phosphate dibasic (K2HPO4) (Wako Pure Chemical Industries, catalog number: 164-04295 )
  21. Potassium phosphate monobasic (KH2PO4) (Wako Pure Chemical Industries, catalog number: 169-04245 )
  22. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Wako Pure Chemical Industries, catalog number: 138-00415 )
  23. Bacto peptone (BD, BactoTM, catalog number: 211677 )
  24. Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 191-01665 )
  25. Bacto agar (BD, BactoTM, catalog number: 214010 )
  26. Distilled water
  27. Cholesterol (Wako Pure Chemical Industries, catalog number: 034-03002 )
  28. 99.5% ethanol (Wako Pure Chemical Industries, catalog number: 057-00456 )
  29. LB broth (BD, Difco, catalog number: 244620 )
  30. Sodium phosphate dibasic (Na2HPO4) (Wako Pure Chemical Industries, catalog number: 194-02875 )
  31. Pentahydrate copper sulphate (CuSO4·5H2O) (Wako Pure Chemical Industries, catalog number: 034-20065 )
  32. Cadmium chloride, anhydrous (CdCl2) (Wako Pure Chemical Industries, catalog number: 036-00125 )
  33. Sodium (mate)arsenite (NaAsO2) (Sigma-Aldrich, catalog number: S7400 )
  34. Gelatin (Sigma-Aldrich, catalog number: G7765 )
  35. Agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500500 )
  36. Tris base (Nacalai Tesque, catalog number: 35434-05 )
  37. Hydrochloric acid (HCl; 35-37%) (Wako Pure Chemical Industries, catalog number: 080-01066 )
  38. SDS (Wako Pure Chemical Industries, catalog number: 191-07145 )
  39. Glycerol (Wako Pure Chemical Industries, catalog number: 075-00616 )
  40. Bromophenol blue (Wako Pure Chemical Industries, catalog number: 021-02911 )
  41. 2-mercaptoethanol (Wako Pure Chemical Industries, catalog number: 135-07522 )
  42. Sodium azide (Wako Pure Chemical Industries, catalog number: 195-11092 )
  43. 1 M CaCl2 (see Recipes)
  44. 1 M K-phosphate buffer (pH 6) (see Recipes)
  45. 1 M MgSO4 (see Recipes)
  46. NGM solution (see Recipes)
  47. 6 cm NGM dish with OP50 (see Recipes)
  48. 0.5% cholesterol (see Recipes)
  49. LB broth (see Recipes)
  50. M9 buffer (see Recipes)
  51. 1 M sodium azide (see Recipes)
  52. 1 M CuSO4 (see Recipes)
  53. 1 M CdCl2 (see Recipes)
  54. 0.5 M sodium arsenite (see Recipes)
  55. 2% gelatin (see Recipes)
  56. 2% agarose (see Recipes)
  57. 2% agarose with 10 mM sodium azide (see Recipes)
  58. Tris-HCl (pH 6.8) (see Recipes)
  59. 1 M 3x SDS sample buffer (see Recipes)

Equipment

  1. Stereomicroscope (Olympus, model: SZ60 )
  2. Centrifuge (TOMY, model: MX-100 )
  3. Rotator (TAITEC, model: RT-50 , catalog number: 0000165-000)
  4. Fluorescent microscope (Nikon Instruments, model: Eclipse E800 ) with highly sensitive camera (ANDOR, model: Zyla 5.5 ) controlled by Nikon NIS-Elements software
  5. Pipetman (P-20, P-200, P-1000; Gilson)
  6. Heat block (TAITEC, model: DTU-Mini , catalog number: 0063287-000)
  7. Autoclave (TOMY DIGITAL BIOLOGY, model: SX-500 )
  8. Microwave oven (Sharp)
  9. Pipettor (Drummond Scientific, model: Pipet-Aid® XP , catalog number: 4-000-101)
  10. Shaker (TAITEC, model: Personal-11 , catalog number: 0000145-000)
  11. Refrigerated incubator (Panasonic Healthcare, model: MIR-154-PJ )
  12. Bunsen burner (Warzef)
  13. Dental burner (Phoenix-Dent, model: APT-3 )

Software

  1. ImageJ software (https://imagej.nih.gov/ij/download.html)
  2. GraphPad Prism QuickCalcs (https://www.graphpad.com/quickcalcs/)

Procedure

  1. Acute reporter assay in liquid
    The acute assay in liquid is used for the determination of the expression of reporter genes induced by short exposure to heavy metals. It can also be used for the preparation of protein extracts of treated worms. For the determination of reporter expression, we recommend using worm strains carrying an appropriate GFP reporter, such as gcs-1p::gfp. Some worm strains carrying a GFP reporter are available from Caenorhabditis Genetics Center (CGC) (Table 1).

    Table 1. CGC strains available for the GFP reporter assay
    Strain in CGC 
    Genotype 
    Organ to determine
    For assay
    LD1171
    ldIs3 [gcs-1p::gfp + rol-6(su1006)]
    Intestine
    As(III) etc. (Wang et al., 2010)
    JF85 
    mtEx60 [numr-1p::gfp + rol-6(su1006)] 
    Intestine
    Cd(II) (Tvermoes et al., 2010)
    JF85 
    mtEx60 [numr-1p::gfp + rol-6(su1006)] 
    Pharynx
    Cu(II) (Tvermoes et al., 2010)

    1. Prepare worms grown on 6 cm NGM dishes with OP50. Although the mixed stage of worms is acceptable, a specific stage of worms (except embryos) can be used if you desire. Avoid starving the worms.
    2. Adding the proper amount of heavy metal solution to M9 buffer (Notes 1 and 2). Dispense 0.5 ml of the solution to 1.5 ml tubes.
    3. Add 1 ml of M9 buffer (without heavy metals) to the NGM dishes, in which worms are grown, and gently shake it.
    4. Transfer the M9 buffer containing worms from NGM dishes to 1.5 ml plastic tubes, using gelatin-coated 1 ml pipetman tips (Note 3). Although the concentration of worms depends on the culture conditions, we usually use approximately 1,000 worms per ml. The worm numbers can be estimated by transferring 10 μl of the solutions to a slide glass just after vortexing and counting the number of worms under a stereomicroscope.
    5. Centrifuge at 700 x g, 2 min, 20 °C. Discard the supernatant.
    6. Add 1 ml M9 buffer to the tube and centrifuge at 700 x g, 2 min. Discard all but a small aliquot (< 0.05 ml) of the supernatant.
    7. Transfer the appropriate amount of worms to 1.5 ml plastic tubes filled with 500 μl of heavy metal-containing M9 buffer. Do not exceed 20 μl of worm volume in each tube.
    8. Rotate (2 sec/rotation to keep an aerobic condition) at 20 °C (or room temperature) by rotator, from 30 min to a few hours.
    9. While waiting, make an agar pad for the end of incubation with heavy metals (Video 1). First, add a double-layered paper tape on the surface of two slide glasses. Place a clean slide glass side by side between the two taped slide glasses (Figure 1A). Put 100 μl of 2% agarose solution with 10 mM sodium azide in the middle of the clean glass (Figure 1B), then immediately put another clean slide glass on the agarose perpendicular to the bottom slide glass (Figure 1C). After 1 min, carefully remove the two taped slide glasses (Figure 1D), and then remove the top slide glass (Figure 1E). Avoid drying until ready to use.

      Video 1. Making an agar pad


      Figure 1. Preparation of agar pad

    10. Centrifuge at 700 x g, 2 min. Discard the supernatant and retain the remaining ~50 μl of M9 with worms.
    11. Transfer the worms from the 1.5 ml tube to an NGM agar dish (without heavy metals) by using a gelatin-coated pipetman tip.
    12. Put 2 μl of M9 on the agarose pad. Pick the worms from the NGM dish, transfer them to the agarose pad, and seal with a cover glass. Immediately use a fluorescent microscope to determine the fluorescence intensity of the reporter gene.

  2. Acute reporter assay on dish
    This assay is suitable for a small number of worms (20-100 worms) in a desired developmental stage, except embryo.
    1. Prepare NGM solution (see Recipes) for dishes.
    2. Add the appropriate amount of heavy metal stock solutions into 50 ml Corning tubes (see Note 2). Add 50 ml of NGM solution at 60 °C into each tube and shake them to mix. Incubate at 60 °C until bubbles disappear.
    3. Pour into 3.5 cm dishes. Pour also the remaining NGM solution into 3.5 cm dishes (use as a control). Leave overnight at room temperature.
    4. Make an overnight culture of OP50 with LB broth. Add 20 μl of the OP50 solution to the 3.5 cm dishes. Leave overnight at room temperature.
    5. Transfer the plates into closed boxes or wrap them with polystyrene bags and store at 4 °C until use.
    6. Just before use, incubate the dishes at 20 °C for 1 h (Note 4).
    7. Put > 20 young adults (grown 1-3 days from late L4 stage) in a 3.5 cm NGM dish containing heavy metal by using a worm pick made from a platinum wire attached to the tip of a Pasteur pipet (Note 5). As a control, use NGM dishes without heavy metals. Incubate for 30 min to several hours at 20 °C.
    8. While waiting, make agarose pads (see Procedure A: Acute reporter assay in liquid). Transfer the worms onto an agarose pad and determine the fluorescence intensity of the reporter gene using a fluorescent microscope.

  3. Acute assay for immunoblotting
    When you want to prepare protein extracts for Immunoblotting, perform the following procedure (Note 6).
    Note: Steps C1 to C8 is the same as A1 to A8 in ‘Acute reporter assay in liquid’.
    1. Centrifuge tubes at 700 x g, 2 min. If the amount of worm pellet seems to exceed 25 μl at this step, divide the samples to additional empty tubes and re-centrifuge them.
    2. Add 1 ml M9 buffer and centrifuge at 700 x g, 2 min. Discard the supernatant and retain the remaining approximately 50 μl of M9 with worms. We estimate the approximate volume of the worm solution by comparing with a sample tube that contains 50 μl of water, and then confirm by sucking the solution with a pipetman adjusted to 50 μl with a gelatin-coated tip.
    3. Add 25 μl of 3x SDS sample buffer.
    4. Boil in water bath (or heat block) for 3 min. Centrifuge the samples at 15,000 x g for 3 min before loading. The samples can be used for immunoblotting of desired proteins, such as PMK-1 and phosphorylated PMK-1 (Inoue et al., 2005).

  4. Growth assay
    Growth assay is used for the determination of the sensitivity to heavy metals. This assay can also be used for the survival assay.
    1. Follow the steps B1-B6 of the procedure in ‘Acute reporter assay on dish’.
    2. Transfer > 20 young adults to a 3.5 cm NGM dish without heavy metals. Incubate for several hours at 20 °C.
    3. Pick ~50 eggs from the NGM dishes and transfer them to an NGM assay plate containing heavy metal (see Video 2). E. coli grown on the normal NGM dish is used as glue to attach the eggs to the platinum wire (Note 7). As a control, transfer the same number of eggs to an NGM plate without heavy metals. Seal the side of each dish with Parafilm. Incubate at 20 °C.
    4. The next day, determine the number of hatched eggs by counting the unhatched eggs. Continue to incubate at 20 °C.
    5. The worms that developed into adulthood are counted 4 days after egg laying (Note 8).

      Video 2. Transfering eggs from a normal to a heavy metal-containing NGM plate

Data analysis

In Acute assays, take pictures by appropriate camera attached to the fluorescent microscope. Then the pictures can be used to quantify the intensity of GFP fluorescence with the ImageJ program. The frequency of growth into adulthood can be calculated by dividing the number of adult animals 4 days after egg laying by the number of eggs hatched on the day after egg laying. The difference between the heavy metal treatment and control data can be tested for statistical significance by Fisher’s or chi-square test using for example GraphPad QuickCalcs.

Notes

  1. Some other researchers prefer to use a different medium (Dong et al., 2004). The difference does not seem to essentially affect the experiments.
  2. The concentrations of heavy metals in the assays vary according to the purpose and the worm strains. The typical concentrations of heavy metals used for the assay are as follows (Table 2):

    Table 2. Typical concentrations of heavy metals for the assays
               
    Acute assays                                   
    Growth assay
    As(III)
    0.5-1 mM (Broeks et al., 1996; Inoue et al., 2005) 
    0.5-1 mM (Broeks et al., 1996; Inoue et al., 2005)
    Cd(II)
    0.1 mM (Dong et al., 2004)
    0.01-0.1 mM (Broeks et al., 1996; Mizuno et al., 2004; Tvermoes et al., 2010)
    Cu(II)
    1 mM (Mizuno et al., 2004)
    0.05-0.1 mM (Broeks et al., 1996; Mizuno et al., 2004; Tvermoes et al., 2010)

    For the initial start-up experiment, we recommend to determine the highest concentration of the heavy metal. We recommend to freshly prepare the assay solution before the assay. The NGM dishes with heavy metals can be stored for several days at 4 °C, avoid both light and drying. We also recommend wearing latex gloves when handling the heavy metal solutions and dishes.
  3. Prepare a gelatin-coated pipetman tip to avoid the sticking the worms inside the tip. Use a pipetman to briefly fill a 1 ml pipette tip with 2% gelatin solution and then quickly empty it.
  4. Avoid the use of wet dishes.
  5. You can change the desired developmental stage of worms for the survival assay to something else than eggs. The viable worms are checked by prodding with a platinum wire.
  6.  The protein extracts are typically used to determine the amount of protein or the phosphorylation level. Usually, at least 100 animals (in the case of the adult stage) are required for immunoblotting.
  7. E. coli OP50 on a 6 cm NGM dish with OP50, which has stayed for several days at room temperature, is best for glue.
  8. Some worms will stick the sidewall of the dish. Subtract the number of the stuck worms from the number of eggs placed on the assay dish in data analysis. 

Recipes

  1. 1 M CaCl2
    Dissolve 147.01 g CaCl2 in dH2O and adjust the volume to 1 L
    Autoclave at 120 °C, 20 min
    Store at room temperature
  2. 1 M K-phosphate buffer (pH 6)
    Dissolve 35.6 g K2HPO4 and 108.3 g KH2PO4 in dH2O
    Adjust pH to 6.0 and the volume to 1 L
    Autoclave at 120 °C, 20 min
    Store at room temperature
  3. 1 M MgSO4
    Dissolve 246.47 g MgSO4·7H2O (or 120.37 g anhydrous MgSO4) in distilled water (dH2O) and adjust the volume to 1 L
    Autoclave at 120 °C, 20 min
    Store at room temperature
  4. NGM solution
    0.75 g Bacto peptone
    0.9 g NaCl
    5.1 g Bacto agar
    0.3 ml cholesterol solution
    Add distilled water to 292 ml
    Autoclave at 120 °C, 20 min
    After autoclaving, add 0.3 ml of 1 M CaCl2, 0.3 ml of 1 M MgSO4 and 7.5 ml of 1 M K-phosphate (pH 6). Keep it at 60 °C
  5. 6 cm NGM dish with OP50
    Pour approximately 10 ml of NGM solution into each Petri dish (60 x 15 mm, Iwaki) and leave at room temperature from several hours to overnight
    Make an overnight culture of OP50 with LB broth
    Add 50 μl of the OP50 solution to each NGM dish
    Leave overnight at room temperature
  6. 0.5% cholesterol
    Dissolve 1 g of cholesterol in 200 ml of 100% ethanol
    Do not autoclave
    Store at room temperature
  7. LB broth
    Dissolve 12.5 g LB broth in 500 ml of dH2O
    Autoclave at 120 °C, 20 min
    Store at room temperature
  8. M9 buffer
    Dissolve 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl in dH2O and adjust the volume to 1 L
    Autoclave at 120 °C, 20 min
    After cooling, add 1 ml of 1 M MgSO4
  9. 1 M sodium azide
    Dissolve 65 mg of sodium azide in 1 ml dH2O
  10. 1 M CuSO4
    Dissolve 25 g CuSO4·5H2O in dH2O and adjust the volume to 100 ml
    Autoclave at 120 °C, 20 min
    Store at room temperature
  11. 1 M CdCl2
    Dissolve 18.3 g CdCl2 in dH2O and adjust the volume to 100 ml
    Autoclave at 120 °C, 20 min
    Store at room temperature
  12. 0.5 M sodium arsenite
    Dissolve 0.65 g NaAsO2 in dH2O and adjust the volume to 10 ml
    Store at -20 °C
  13. 2% gelatin
    Dissolve 2 g gelatin in 100 ml of dH2O
    Autoclave at 120 °C, 20 min
    Store at room temperature
  14. 2% agarose
    Add 2 g agarose (UltraPure agarose, Invitrogen) to 100 ml of dH2O
    Dissolve by heating using a microwave oven
    Incubate at 60 °C until ready to use
    Stable for several days at 60 °C
    Keep at room temperature for longer storage (heating is required just prior to use)
  15. 2% agarose with 10 mM sodium azide
    Add 1 ml of melted 2% agarose into a 1.5 ml Eppendorf tube
    Immediately place it in a heat block and keep it at 60 °C
    Then add 10 μl of 1 M sodium azide and vortex it. Use it immediately
  16. 1 M Tris-HCl (pH 6.8)
    18.17 g Tris base
    700 ml H2O
    Approx.120 ml HCl
    Adjust pH by adding HCl
    Check the pH by a pH probe
    Add distilled H2O to adjust to 1 L
  17. 3x SDS sample buffer
    3.75 ml of 0.5 M Tris-HCl (pH 6.8)
    0.6 g SDS
    3 ml glycerol
    1.5 mg bromophenol blue
    Add distilled water to 8.5 ml
    Store at room temperature, avoid light
    Add 0.15 ml of 2-mercaptoethanol to 0.85 ml of the buffer just prior to use

Acknowledgments

This work was supported by grants from the Ministry of Education, Culture and Science of Japan. Many other researchers have utilized similar protocols to test sensitivity to heavy metals. The protocols are based on the way that we measure heavy metal sensitivity in our lab. This protocol is essentially adopted previous works (Mizuno et al., 2004; Inoue et al., 2005).

References

  1. Broeks, A., Gerrard, B., Allikmets, R., Dean, M. and Plasterk, R. H. (1996). Homologues of the human multidrug resistance genes MRP and MDR contribute to heavy metal resistance in the soil nematode Caenorhabditis elegans. EMBO J 15(22): 6132-6143.
  2. Dong, J., Song, M. O. and Freedman, J. H. (2004). Identification and characterization of a family of Caenorhabditis elegans genes that is homologous to the cadmium-responsive gene cdr-1. Biochim Biophy Acta 1727: 16-26.
  3. Green, M. R. and Sambrook, J. (2012). Molecular cloning: a laboratory manual (4th edition). Cold Spring Harbor Laboratory Press.
  4. Inoue, H., Hisamoto, N., An, J. H., Oliveira, R. P., Nishida, E., Blackwell, T. K. and Matsumoto, K. (2005). The C. elegans p38 MAPK pathway regulates nuclear localization of the transcription factor SKN-1 in oxidative stress response. Genes Dev 19(19): 2278-2283.
  5. Mizuno, T., Hisamoto, N., Terada, T., Kondo, T., Adachi, M., Nishida, E., Kim, D. H., Ausubel, F. M. and Matsumoto, K. (2004). The Caenorhabditis elegans MAPK phosphatase VHP-1 mediates a novel JNK-like signaling pathway in stress response. EMBO J 23(11): 2226-2234.
  6. Schwartz, M. S., Benci, J. L., Selote, D. S., Sharma, A. K., Chen, A. G., Dang, H., Fares, H. and Vatamaniuk, O. K. (2010). Detoxification of multiple heavy metals by a half-molecule ABC transporter, HMT-1, and coelomocytes of Caenorhabditis elegans. PLoS One 5(3): e9564.
  7. Tvermoes, B. E., Boyd, W. A. and Freedman, J. H. (2010). Molecular characterization of numr-1 and numr-2: genes that increase both resistance to metal-induced stress and lifespan in Caenorhabditis elegans. J Cell Sci 123(Pt 12): 2124-2134.
  8. Wang, J., Robida-Stubbs, S., Tullet, J. M., Rual, J. F., Vidal, M. and Blackwell, T. K. (2010). RNAi screening implicates a SKN-1-dependent transcriptional response in stress resistance and longevity deriving from translation inhibition. PLoS Genet 6(8).
  9. Valko, M., Morris, H. and Cronin, M. T. (2005). Metals, toxicity and oxidative stress. Curr Med Chem 12(10): 1161-1208.

简介

生物开发了许多保护系统,以减少重金属的毒性。线虫秀丽隐杆线虫已广泛用于确定重金属的保护机制。可以通过报告基因的表达来监测对重金属的反应,而敏感性可以通过量化暴露于重金属后的生长或存活率来确定。

背景 一些重金属如砷,镉和汞已知对包括人类在内的大多数生物有害(Valko et al。,2005)。为了降低这些金属的毒性,生物开发出各种保护系统。线虫秀丽隐杆线虫已被用于了解重金属的保护机制。以前的研究表明,许多基因,如解毒酶,转录因子和信号传导因子都参与了该生物体中重金属的保护(Broeks et al。,1996; Mizuno et al。 ,2004; Inoue等人,2005; Schwartz等人,2010)。除了测量报告基因表达外,生存力和生长的测定通常用于监测重金属在C中的影响。线虫。在本协议中,我们描述了使用C测定砷,铜和镉的方法。线虫。

关键字:秀丽隐杆线虫, 砷, 镉, 铜

材料和试剂

  1. 0.2-10μl移液器吸头,Corning ® Isotip ®过滤(Corning,目录号:4807)
  2. 1-200μl移液器吸头(Corning,Axyge ®,目录号:TF-200-R-S)
  3. 100-1,000μl移液器吸头(Corning,Axygen ®,目录号:TF-1000-R-S)
  4. 1.5ml微量离心管(RNase,DNase和无热原)(Corning,Axygen,目录号:MCT-150-C)
  5. 0.2ml薄壁PCR管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3412)
  6. Falcon ®将50ml锥形离心管(Corning,Falcon ®,目录号:352070)
  7. pSuper基本载体(Oligoengine,目录号:VEC-pBS-0002)
  8. miRNA编码寡核苷酸(PAGE纯化)5'-pGATCTAGCACGACTCGCAGCTCCCAAGAGCCTAACCCGTGGATTTAAACGGTAAACATCACAAGTTAGGGTCTCAGGGACTGAGAGGAGCGCAA-3'
  9. 用于最小H1(mH1)启动子的寡核苷酸(PAGE纯化)
    mH1-Fw:5'-pAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGA-3'
    mH1-Rv:5'-pGATCTCTGTGCTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATG-3'
  10. UltraPure TM DNase / RNase-Free蒸馏水(Thermo Fisher Scientific,Invitrogen TM,目录号:10977015)
  11. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2670-100G)
  12. dNTP设置100mM溶液(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0181)
  13. 用于mH1启动子扩增的寡核苷酸引物(HPCL纯化)
    NbBpu-Fw
    5'-pTTAGGAGTTTTCTCCTAAGCATATTTGCATGTCGCTATGTGTTCTG-3'
    BamHI-Rv
    5'-TGCAGGATCCCTGTGCTCTCATACAGAACTTATAAGATTCCC-3'
  14. DNA聚合酶,重组体(5U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EP0402)
  15. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28106)
  16. 10x FD缓冲液(Thermo Fisher Scientific,Thermo Scientific TM,目录号:B64)
  17. Nb.Bpu10I(5U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:ER1681)
  18. FastDigest Bam(Thermo Fisher Scientific,Thermo Scientific TM,目录号:FD0054)
  19. shRNA编码寡核苷酸(PAGE纯化)5'-pGATCTAAAAAGAGCTGTTTCTGAGGAGCCTCTCTTGAAGGCTCCTCAGAAACAGCTCTTTTTA-3'
  20. T4 DNA连接酶(5U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EL0014)
  21. 中和寡核苷酸(HPLC纯化)
    5'-TTAGGAGTTTTCTCCTAA-3'
  22. 腺苷5'-三磷酸二钠盐水合物(Sigma-Aldrich,目录号:A2383-1G)
  23. FastDigest II(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:FD0083)
  24. T7 DNA聚合酶(10U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EP0081)
  25. 苯酚溶液(Sigma-Aldrich,目录号:P4557-100ML)
  26. 氯仿(Sigma-Aldrich,目录号:288306-1L)
  27. 3-甲基-1-丁醇(Sigma-Aldrich,目录号:309435-100ML)
  28. 乙醇,绝对(Fisher Scientific,目录号:BP28184)
  29. 3M醋酸钾(pH 4.8)
  30. 乙酸钠(Sigma-Aldrich,目录号:S2889-250G)
  31. FastDigest RI(Thermo Fisher Scientific,Thermo Scientific TM,目录号:FD0274)
  32. 琼脂糖,LE,分析纯(Promega,目录号:V3125)
  33. 溴化乙锭溶液(Bio-Rad Laboratories,目录号:1610433)
  34. GeneRuler DNA梯形混合物(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:SM0331)
  35. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888-500G)
  36. Tris-HCl(Powder)(Roche Diagnostics,目录号:10812846001)
  37. EDTA(Sigma-Aldrich,目录号:EDS-100G)
  38. 10x杂交缓冲液(参见食谱)
  39. TE缓冲(见配方)

设备

  1. 移液器(Gilson,PIPETMAN ®经典型号:P2,P20N,P200N和P1000N)
  2. 标准热循环仪(Thermo Fisher Scientific,Applied Biosystems TM,型号:GeneAmp PCR System 9700)
    注意:本产品已停产。
  3. Gel doc(Bio-Rad Gel Doc Imager)
  4. 凝胶运行装置(Thermo Fisher Scientific,Amersham Biosciences TM
  5. 凝胶染色托盘
  6. 台式离心机(Eppendorf,型号:5430 R)
  7. 热块(Eppendorf,型号:Thermomixer ®舒适)
  8. 分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop 2000)
  9. 玻璃烧杯(Schott,Duran)
  10. 微波(松下)

软件

  1. ImageJ( http://imagej.nih.gov/ij/

程序

在本协议中,我们描述了a)shRNA(例如db-iPRT-hp-s / as:靶向荧光素酶)或b)miRNA(例如db-hp-miR-125b-1:表达has-miR- 125b-1)表达最小化的哑铃载体(Jiang等人,2016)。对于这些哑铃,shRNA或miRNA表达由人类最小H1(mH1)启动子驱动。使用引入用于切口内切核酸酶的切割位点的正向引物和引入常规限制性内切核酸酶切割位点的反向引物对mH1启动子进行PCR扩增。在PCR产物与内切核酸酶两者孵育后,通过切口反应产生的重折叠突出形成哑铃的一个环结构,而另一个环通过连接shRNA或miRNA编码的发夹寡脱氧核糖核苷酸形成。在适当的限制酶存在下进行连接,以抑制产生不必要的产物(Cost,2007)。通过核酸酶(ELAN)方法辅助的酶促连接抑制了在限制性内切核酸酶存在下被切割的环寡聚体或表达盒组成的失调产物如二聚体的形成(在该实施例中, HI和Bgl II),从而有助于形成不包含相应的内切核酸酶切割位点的预期哑铃结构。最后,通过外切核酸酶处理破坏未连接的DNA,并纯化外切核酸酶哑铃(图2)。


图2.表达小RNA的最小化哑铃载体的生产策略。该方案包括以下步骤:首先,通过PCR扩增mH1启动子序列。通过PCR引物引入适当的切口酶(NE)和常规限制(RE)位点。第二,使用相应的切口和限制酶消化扩增的启动子DNA。第三,将经消化的DNA纯化,并用T4DNA连接酶进行第四次退火并与发夹DNA模板寡核苷酸连接,形成shRNA(左侧)或miRNA(右侧)表达哑铃载体。添加中和寡核苷酸和柱纯化步骤显着提高了哑铃产量。最后,通过外切核酸酶处理除去未连接的DNA,并使用标准DNA纯化技术纯化哑铃载体DNA(Jiang等人,2016)。

  1. 寡核苷酸退火
    1. 通过在10μl1x杂交缓冲液中加热500pmol寡核苷酸至95℃3分钟(Thermomixer Comfort,Eppendorf)并使溶液在1小时内冷却至室温来实现sh / miRNA编码DNA发夹的正确折叠。
    2. 该方案中的mH1启动子已经通过引入反向转录终止子而被修饰。因此,将寡核苷酸mH1-Fw和mH1-Rv溶于无核酸酶的蒸馏水中至浓度为100μM。通过加热至95℃3分钟(Thermomixer Comfort,Eppendorf),在10μl1x杂交缓冲液中退火500pmol,然后在1小时内将溶液冷却至室温(注1)。

  2. mH1启动子序列的PCR扩增
    1. 将以下组件添加到薄壁PCR管中:


    2. 使用以下热循环仪条件进行PCR:


      按照制造商的建议,使用QIAquick PCR Purification Kit(QIAGEN)纯化PCR产物。&nbsp;

  3. PCR产物的内切核酸切割
    1. 在室温下按照所示顺序合并反应组分:


    2. 将反应混合物在37℃孵育4小时,然后在85℃孵育5分钟以灭活酶(注2)。&nbsp;
    3. 然后根据制造商的建议(注3),用PCR QIAquick PCR Purification Kit(QIAGEN)纯化消化的DNA。
      &nbsp;
  4. mH1启动子和sh / miRNA编码的发夹寡核苷酸的退火和连接
    1. 启动子DNA和sh / miRNA编码发夹寡核苷酸的退火是通过将1x杂交缓冲液中的每种DNA等摩尔量(80pmol)加热至95℃3分钟(Thermomixer Comfort,Eppendorf)并使溶液冷却至室1小时内温度。
    2. 退火后,连接反应按照指示顺序设置(注4):


    3. 将反应混合物在22℃孵育4小时或过夜,然后在80℃下孵育10分钟以灭活酶(注5)。
    4. 从反应混合物中取出样品(1-2μl)进行凝胶电泳分析
  5. 外来核酸酶处理和纯化哑铃形载体
    1. 在上述200μl连接混合物中加入1μlT7 DNA聚合酶(10 U /μl),37℃孵育1 h,然后在80°C孵育10 min,使酶失活(注6)。
    2. 从反应混合物中取出样品(1-2μl)进行凝胶电泳分析
    3. 对取出的样品进行分析性1.5%琼脂糖凝胶电泳,以监测哑铃载体DNA的转化产率和纯度(图3)。
    4. 使用标准苯酚/氯仿提取纯化哑铃DNA,然后乙醇沉淀。详细地,将等体积的苯酚/氯仿/异戊醇(25:24:1)加入到水性哑铃溶液中,旋涡30秒,并通过离心(5分钟,13,000xg)分离水相和有机相)。将上层水相转移到新的Eppendorf管中,并重新提取苯酚残留物。因此,加入等体积的氯仿/异戊醇(24:1),用手严格摇动30秒,并通过离心(30秒,13,000×g / g)分离相。将上层水相转移到新的Eppendorf管中,并重复两次重萃取过程(注7)。对于乙醇沉淀,用蒸馏水将水相加至400μl,然后加入0.1体积(40μl)3M乙酸钾(pH 4.8),然后加入2.5体积(1,100μl)乙醇,将溶液混合,在-20℃下孵育20分钟。通过以13,000 x g,4℃离心15分钟使DNA沉淀。然后将颗粒风干。将纯化的DNA溶解在TE缓冲液或蒸馏水中

      图3.不同处理后哑铃载体DNA的分析凝胶电泳。其他处理增加了哑铃转换产率:基本方案(1),中和寡聚(50 pmol)的基本方案(2),基本方案与柱纯化(使用QIAgen PCR纯化试剂盒和QIAgen的以下标准方案)(3)和两种处理的基本方案(4)。中和寡聚体结合在切割酶切割后释放的寡核苷酸,并防止这可能再次结合到产生的突出端。柱纯化除去小切割产物并防止它们在随后的连接步骤中降级。通过另外的柱纯化步骤实现了最高的转化率(91%)。转化产率定义为当比较核酸外切酶处理前后预期哑铃的大小时获得的产量。 CP,柱纯化; Neu,中和寡核苷酸(Jiang等人,2016)。

数据分析

哑铃DNA转化产率分析:

  1. 使用魔杖工具使用ImageJ测量电泳凝胶中与哑铃产品相对应的DNA条带的强度。
  2. 通过将外切核酸酶处理后的条带的强度除以连接样品中相应条带的强度来计算转化产率。

笔记

  1. 或者,可以通过从质粒DNA pSuper-mH1的PCR扩增产生mH1序列,其通过将杂交的寡核苷酸克隆到pSuper碱性质粒中构建。为了将退火的序列插入pSuper碱性质粒,首先用EcoRI和BglⅡ在37℃下消化载体(1μg)4小时。消化后,使用QIAgen PCR Purification Kit纯化DNA。使用等摩尔量(0.25pmol)消化的载体骨架进行连接,并在22℃退火插入物4小时。通过测序确认正确的克隆。如果使用pSuper-mH1进行PCR反应,则使用10ng作为模板
  2. 根据Taki等人先前的报告,我们使用Nb.Bpu10I作为切口酶。 (2004年)。酶供应10倍缓冲液R,但是在咨询了赛默飞世尔技术支持后,使用通用的10倍缓冲液FD。
  3. 虽然这一步骤的消化产物可以直接用于连接,我们发现一个额外的纯化步骤可以大大提高哑铃转化率,如图2所示。
  4. 在一些情况下,将中和寡核苷酸加入到连接反应中以抑制短切片段的再退火(参见图2)。在用1mM最终ATP补充的FD缓冲液中进行连接,而不是连接缓冲液,因为包括连接酶的所有酶在该缓冲液中为100%活性。为了连接sh / miRNA编码的发夹模板和启动子DNA,我们按照如前所述的核酸酶(ELAN)技术进行酶连接辅助(Cost,2007)。将sh / miRNA编码的发夹寡核苷酸序列PAGE纯化,可直接用于连接
  5. 根据我们的经验,连接反应在4小时内完成,较长的孵育时间没有提高哑铃产量。
  6. T7 DNA聚合酶在FD缓冲液中显示出100%的活性,因此直接加入到连接混合物中
  7. 必须用手摇动,而不是旋涡,以便有效地混合相位。

食谱

  1. 10x杂交缓冲液
    1 M NaCl
    100mM MgCl 2
    200mM Tris-HCl,pH7.4
  2. TE缓冲区
    10mM Tris-HCl,pH8.0
    1 mM EDTA

致谢

这里描述的协议在以前在Jiang等人开发和使用。 (2016)。新加坡国立大学新加坡国立医科大学附属新加坡国立大学新加坡国立医学研究委员会(新加坡国立医学研究理事会,新加坡国立医科大学核医学研究中心,新加坡国立医科大学,尼日利亚国立大学,尼日利亚共和国),以及教育部新加坡[学术研究基金(AcRF)第1级教师研究委员会(FRC)授予T1-2011Sep-04和T1-2014Apr-02以及“基础科学研究种子基金”号T1-BSRG 2015-05]。作者宣称相互竞争的经济利益。涵盖主要工作的专利申请正在等待。

参考

  1. Chen,ZY,He,CY,Meuse,L.和Kay,MA(2004)。通过质粒细菌DNA元件在体内沉默附加型转基因 Gene Ther 11(10):856-864。
  2. Chen,ZY,Riu,E.,He,CY,Xu,H. and Kay,MA(2008)。通过质粒细菌骨架DNA沉默肝脏中的附加型转基因表达与CpG甲基化无关。Mol Ther 16(3):548 -556。
  3. 成本,GJ(2007)。由核酸酶辅助的酶结合:同时连接和消化促进DNA的有序组装。 Nat Protoc 2(9):2198-2202。
  4. 江泽民,俞,H.,曹,谭,谭,GS,Goh,SC,Patel,P.,Chua,YK,Hameed,NB,Bertoletti,A。和Patzel,V。(2016) 哑铃型遗传最小载体的高级设计改善了非编码和编码RNA表达。 Mol Ther 24(9):1581-1591。
  5. Nicol,F.,Wong,M.,MacLaughlin,FC,Perrard,J.,Wilson,E.,Nordstrom,JL和Smith,LC(2002)。&lt; a class =“ke-insertfile”href =“http ://www.ncbi.nlm.nih.gov/pubmed/12365000“target =”_ blank“>阴离子聚合物聚-L-谷氨酸增强体内肌内注射传递的质粒的转基因表达, em> electroporation。 Gene Ther 9(20):1351-1358。
  6. Schakowski,F.,Gorschluter,M.,Buttgereit,P.,Marten,A.,Lilienfeld-Toal,MV,Junghans,C.,Schroff,M.,Konig-Merediz,SA,Ziske,C.,Strehl,J 。,Sauerbruch,T.,Wittig,B.和Schmidt-Wolf,IG(2007)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed / 17354609“target =”_ blank“>最小尺寸MIDGE载体在体内改善转基因表达。在体内 21(1):17-23。
  7. Schakowski,F.,Gorschluter,M.,Junghans,C.,Schroff,M.,Buttgereit,P.,Ziske,C.,Schottker,B.,Konig-Merediz,SA,Sauerbruch,T.,Wittig,和Schmidt-Wolf,IG(2001)。一本小说最小尺寸载体(MIDGE)改善结肠癌细胞中的转基因表达,并避免转染不需要的DNA。 Mol Ther 3(5 Pt 1):793-800。
  8. Taki,M.,Kato,Y.,Miyagishi,M.,Takagi,Y.和Taira,K。(2004)。基于有效构建的哑铃形DNA的细胞中的小干扰RNA表达。 Angew Chem Int Ed Engl 43(24):3160-3163
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Pastuhov, S. I., Shimizu, T. and Hisamoto, N. (2017). Heavy Metal Stress Assay of Caenorhabditis elegans. Bio-protocol 7(11): e2312. DOI: 10.21769/BioProtoc.2312.
  2. Pastuhov, S. I., Fujiki, K., Tsuge, A., Asai, K., Ishikawa, S., Hirose, K., Matsumoto, K. and Hisamoto, N. (2016). The Core Molecular Machinery Used for Engulfment of Apoptotic Cells Regulates the JNK Pathway Mediating Axon Regeneration in Caenorhabditis elegans. J Neurosci 36(37): 9710-9721.
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