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Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants
秀丽隐杆线虫肿瘤生殖细胞系突变体生殖细胞的原代培养系统   

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Abstract

The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al., 2016.

Keywords: C. elegans(秀丽隐杆线虫), Primary culture(原代培养), Germ stem cells(生殖干细胞), Tissue culture(组织培养)

Background

C. elegans hermaphrodite germ cells are generated in two adult stem cell niches located in the distal regions of the two gonad arms (Hansen and Schedl, 2013; Kimble and Seidel, 2013). In wild-type hermaphrodites, mitotic germ cells are restricted to the distal, stem cell niche regions of the gonad arms. Wild-type germ cells are syncytial, and contain an opening to a common cytoplasm that extends through the central region of the gonad arms. C. elegans tumorous germline mutants have increased mitotic proliferation of germ cells throughout the gonad. We discovered that tumorous germline mutants generally have fully cellularized germ cells that contain intact plasma membranes (Chaudhari et al., 2016). This cellularization allows the isolation of the germ cells and their maintenance in culture. This protocol describes the methodology and tissue culture medium to isolate and maintain germ cells from tumorous mutants in culture. While a culture medium has been described for the primary culture of C. elegans embryonic and larval cells (Strange et al., 2007; Zhang and Kuhn, 2013), germ cells do not survive in this medium (Chaudhari et al., 2016). We created a culture medium for germ cells that is called CeM1 for ‘C. elegans medium 1’. We anticipate that other iterations of the medium could be given subsequent numbers, e.g., ‘CeM2’. This protocol, first reported in Chaudhari et al., 2016, allows the isolation of essentially pure populations of germ cells and their maintenance in in vitro primary cultures. This culture system can allow new experimental approaches to probe germ cell biology in C. elegans.

Materials and Reagents

  1. Polystyrene tubes, 15 ml (Corning, Falcon®, catalog number: 352099 )
  2. Polypropylene tubes, 15 ml (Corning, catalog number: 25319-15 )
  3. Polypropylene tubes, 50 ml (Corning, catalog number: 25330-50 )
  4. Filter pipette tips, 100-1,000 µl (Corning, catalog number: 4809 )
  5. Filter pipette tips, 20-200 µl (Fisher Scientific, catalog number: 02-707-430 )
  6. Filter pipette tips, 2-20 µl (Fisher Scientific, catalog number: 02-707-435 )
  7. Filter pipette tips, 0.1-10 µl (Fisher Scientific, catalog number: 02-707-439 )
  8. Filter units 150 ml, PES 0.22 µm (EMD Millipore, catalog number: SCGPU01RE )
  9. Filter units 500 ml, PES 0.22 µm (EMD Millipore, catalog number: SCGPU05RE )
  10. Aluminum foil
  11. Tissue culture dish, 35 x 10 mm (Corning, Falcon®, catalog number: 353001 )
  12. Tissue culture dish, 24-well (Corning, catalog number: 3524 )
  13. Tissue culture dish, 96-well (Corning, catalog number: 3596 )
  14. Cell scrapers, 39 cm, disposable (SARSTEDT, catalog number: 83.1831 )
  15. Serological pipets, disposable 10 ml (Fisher Scientific, catalog number: 13-678-11E )
  16. Tissue culture flask, 12.5 cm2 (Corning, Falcon®, catalog number: 353018 )
  17. Platinum wire, 0.25 mm (Alfa Aesar, catalog number: 10288 )
  18. Needles, 21 G x 1 ½ (BD, catalog number: 305167 )
  19. Parafilm
  20. 0.5 ml centrifuge tube
  21. Microcentrifuge tubes, 1.5 ml (Fisher Scientific, catalog number: 05-408-129 )
  22. Razor blades, single edge
  23. Paper towel
  24. C. elegans tumorous germline mutant strain ET507, daf-16(mu86) I; cki-2(ok2105) II; glp-1(ar202) III
  25. Escherichia coli strain OP50
    Notes:
    1. We did not perform tests to determine how materials and reagents from other manufacturers function in germ cell isolation and culture, with the exception of the use of STARSTEDT 96-well plates (non-tissue culture-treated) (STARSTEDT, catalog number: 82.1581.001 ) for the in vitro culture of germ cells, which resulted in the premature death of the germ cells.
    2. C. elegans germline tumor mutant strain ET507 (as well as other tumorous mutant strains) and E. coli bacteria OP50 are available from the Caenorhabditis Genetics Center (CGC), http://www.cgc.cbs.umn.edu.
  26. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11550 )
  27. Amberlite IRA 400-CL (Sigma-Aldrich, catalog number: 247669 )
  28. Charcoal-dextran (Sigma-Aldrich, catalog number: C6241 )
  29. Liquid nitrogen
  30. Phosphate buffered saline (PBS) (GE Healthcare, HyCloneTM, catalog number: SH30256.01 )
  31. Schneider’s insect medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21720024 )
  32. Leibovitz’s L-15 medium without phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 21083027 )
  33. Penicillin/streptomycin (Sigma-Aldrich, catalog number: P4333 )
  34. Hemin chloride (MP Biomedicals, catalog number: 0219402501 )
  35. RPMI Vitamins (Sigma-Aldrich, catalog number: R7256 )
  36. L-glutathione, reduced (Sigma-Aldrich, catalog number: G4251 )
  37. Normocin (InvivoGen, catalog number: ant-nr-1 )
  38. Trehalose (Sigma-Aldrich, catalog number: T0167 )
  39. Osmolality standard, 100 mmol/kg (Wescor, catalog number: OA-010 )
  40. Osmolality standard, 290 mmol/kg (Wescor, catalog number: OA-029 )
  41. Osmolality standard, 1,000 mmol/kg (Wescor, catalog number: OA-100 )
  42. Water, molecular biology grade (GE Healthcare, HyCloneTM, catalog number: SH30538.02 )
  43. Tryptone (Fisher Scientific, catalog number: BP1421-500 )
  44. Yeast extract (Fisher Scientific, catalog number: BP9727-2 )
  45. Sodium chloride (NaCl) (Avantor Performance Materials, J.T. Baker®, catalog number: 3624-05 )
  46. Sodium hydroxide (NaOH) (Avantor Performance Materials, J.T. Baker®, catalog number: 3728-01 )
  47. Bacto-peptone (BD, BactoTM, catalog number: 211677 )
  48. Agar (RPI, catalog number: A20020-5000 )
  49. Cholesterol (Avantor Performance Materials, J.T. Baker®, catalog number: 1580-01 )
  50. Ethanol, 100% (used to make 70% with distilled water) (Decon Labs, catalog number: 2716 )
  51. Magnesium sulfate, anhydrous (MgSO4) (Avantor Performance Materials, J.T. Baker®, catalog number: 2506-01 )
  52. Calcium chloride (CaCl2) (Avantor Performance Materials, J.T. Baker®, catalog number: 1313-01 )
  53. Potassium phosphate, monobasic (KH2PO4) (Avantor Performance Materials, J.T. Baker®, catalog number: 3246-05 )
  54. Sodium phosphate, heptahydrate (Na2HPO4·7H2O) (Fisher Scientific, catalog number: S373-3 )
  55. Sodium hypochlorite, 6% (RICCA Chemical, catalog number: 7495.7-32 )
  56. Tetracycline (Sigma-Aldrich, catalog number: 87128 )
  57. Chloramphenicol (RPI, catalog number: C61000-25.0 )
  58. Kanamycin (Fisher Scientific, catalog number: BP906-5 )
  59. Ethidium homodimer (Biotium, catalog number: 40010 )
  60. Dimethyl sulfoxide (DMSO) (Fisher Scientific, catalog number: D128-500 )
  61. Hoechst 33342 (Sigma-Aldrich, catalog number: B2261 )
  62. Calcein-AM (Biotium, catalog number: 80011 )
  63. 2xYT bacterial medium (see Recipes)
  64. LB bacterial medium (see Recipes)
  65. 3x NGM agar plates seeded with OP50 bacteria (see Recipes)
  66. M9 buffer (see Recipes)
  67. Sodium hypochlorite solution (see Recipes)
  68. Platinum-wire worm pick (see Recipes)
  69. Tetracycline stock (see Recipes)
  70. Chloramphenicol stock (see Recipes)
  71. Kanamycin stock (see Recipes)
  72. Cholesterol stock (see Recipes)
  73. Antibiotic-enriched PBS with heat-killed bacteria (see Recipes)
  74. Hemin chloride stock (see Recipes)
  75. Stock of Hoechst 33342, calcein-AM, and ethidium homodimer (see Recipes)

Equipment

  1. Temperature-controlled water bath
  2. Table-top centrifuge with swinging bucket rotor for 15 ml and 50 ml tubes
  3. Rotator, single speed (Barnstead Thermolyne, catalog number: C415110 )
  4. 500 ml plastic bottle
  5. Freezing point osmometer (Advanced Digimatic Osmometer 3DII, Advanced Instruments, model: Model 3D2 )
  6. Low-temperature incubator
  7. Bottom illuminated stereomicroscope with frosted glass stage or frosted mirror (various Nikon or Leica models)
  8. Airtight containers (LockandLock, various size Lock & Lock containers available from Amazon)
  9. Phase-contrast inverted compound microscope (various Nikon, Leica, or Olympus models with 10x and 20x objectives)
  10. Nutator (BD, catalog number: 421105 )
  11. Hemacytometer (Reichert Bright-Line, Hauser Scientific, catalog number: 1492 )
  12. Upright fluorescence compound microscope (various Nikon, Leica, or Olympus models with 5x, 20x, 40x, and 64x objectives)
  13. 2 L Erlenmeyer flasks (Corning, PYREX®, catalog number: 4980-2L )
  14. Autoclave
  15. 50 ml Erlenmeyer flasks (Corning, PYREX®, catalog number: 4980-50 )
  16. Plastic 1 L measuring cup with handle (Fisher Scientific, catalog number: 02-543-36C )
  17. Stir bar
  18. Bottles, 125 ml, glass (WHEATON, catalog number: 219815 )
  19. Bunsen burner
  20. Analytical balance
  21. pH meter
  22. Pipette controller (Pipet-Aid) (Drummond Scientific, catalog number: 4-000-110 )
  23. Pasteur pipets, 9 inch, glass (Fisher Scientific, catalog number: 22-063172 )
  24. Bulbs for Pasteur pipets (Fisher Scientific, catalog number: 03-448-21 )
  25. Laminar-flow tissue culture hood
  26. Aspirator

Procedure

  1. Pretreatment of FBS
    1. If the FBS is stored frozen in aliquots (at -20 °C or -80 °C), thaw the appropriate amount of FBS to make the desired volume of CeM1 medium (FBS is used at a final concentration of 8%). Note, pretreatment of FBS leads to a loss of ~20% of the volume due to trapping of FBS in charcoal-dextran, e.g., 25 ml of FBS will produce ~20 ml of pre-treated FBS. FBS can be thawed at room temperature or in a 37 °C water bath. The pretreatment contains two overnight incubation steps. The incubation of FBS with Amberlite IRA 400-CL beads and charcoal-dextran removes hydrophobic molecules, such as steroid hormones. The pretreatment steps are required to maintain germ cell viability in the CeM1 medium (Chaudhari et al., 2016). It is not known how the pretreatments alter the FBS to improve cell survival.
    2. Heat inactivate the thawed FBS by incubation at 56-65 °C for 30 min in a temperature-controlled water bath with occasional swirling (every 5-10 min).
    3. Prewash an amount of Amberlite IRA 400-CL beads that is based on a final concentration of 50 mg of beads per ml of FBS that will be subject to pretreatment; for 25 ml of FBS, this would be 1.25 g of beads. For the prewash, place the beads in a 15 ml or 50 ml polypropylene tube. The beads are prewashed by adding molecular-grade water to the tube (~13 or ~45 ml depending on whether a 15 ml or 50 ml tube is used) and inverting the tube several times. The beads are collected by centrifugation at 930 x g for 1 min in a table-top centrifuge. Aspirate the water above the beads. Repeat the wash two times. Remove residual water between the beads by placing a 1,000 µl pipet tip against the bottom of the tube and pipetting out the water.
    4. Add the heat-inactivated FBS to the tube with the washed Amberlite IRA 400-CL beads.
    5. Rotate the FBS and Amberlite IRA 400-CL beads for 4-6 h at room temperature in a single-speed rotator.
    6. Pre-wash a second set of Amberlite IRA 400-CL beads (1.25 g for 25 ml of FBS), as was done in step A3.
    7. Spin down the beads (930 x g for 1 min), and transfer the FBS to a second batch of washed Amberlite IRA 400-CL beads in a second tube.
    8. Rotate the FBS and beads overnight at 4 °C in the rotator.
    9. The next day, prepare charcoal-dextran for incubation with the FBS. The amount of charcoal-dextran is based on a final concentration of 100 mg of charcoal-dextran per ml of FBS; for 25 ml of FBS, this would be 2.5 g of charcoal-dextran. Precrush the charcoal-dextran against the side of the weigh dish–to break-up clumps. Place in a 15 ml or 50 ml polypropylene tube.
    10. Spin down the Amberlite IRA 400-CL beads (930 x g for 1 min), and transfer the FBS to the tube with the charcoal-dextran.
    11. Rotate the FBS with the charcoal-dextran overnight at 4 °C in the rotator.
    12. Remove the charcoal-dextran with two sequential centrifugations, each at 2,850 x g for 30 min, 4 °C, followed by transferring FBS to a new polypropylene tube (leaving a small fraction, ~100 µl, over the pellet to ensure that no residue from the pellet is taken).
    13. FBS that has been treated by heat-inactivation, and incubations with Amberlite IRA 400-CL and charcoal-dextran can either be used immediately, stored at 4 °C for one to two weeks, or stored for longer periods (months or a few years) at -80 °C (after snap-freezing aliquots in liquid nitrogen).

      Notes:

      1. Different lots of FBS have large effects on the long-term survival of germ cells in culture. It would be advantageous to obtain samples of multiple lots from FBS manufacturers/distributors to test for germ cell survival prior to purchasing a specific lot of FBS.
      2. Throughout the protocol, the centrifuge speeds are based on a Beckman Coulter Allegra X-15R table-top centrifuge with a swinging bucket rotor, which has the following rpm to rcf (x g) conversions: 300 rpm = 21 x g; 1,000 rpm = 230 x g; 2,000 rpm = 930 x g; 3,500 rpm = 2,850 x g; 4,000 rpm = 3,700 x g.

  2. Preparation of CeM1 medium
    1. Prepare CeM1 medium in a bottle that has not been washed with soap (e.g., a 500 ml plastic bottle that previously held water, PBS, or tissue culture medium, and that was cleaned by rinsing with distilled water and air drying). If less than 50 ml of medium is prepared, it can be made in a 50 ml polypropylene tube.
    2. The percentages/concentrations of CeM1 ingredients are described in Table 1. Initially, add Schneider’s insect medium, Leibovitz’s L-15 medium without phenol red, pretreated FBS, penicillin/streptomycin, RPMI vitamins, and normocin.

      Table 1. CeM1 medium components


    3. Add cholesterol from a 10 mg/ml stock in ethanol (see Recipes).
    4. Add trehalose at 18 mg/ml, and swirl the bottle to dissolve the powder. This should produce an osmolality that is lower than the desired 390 mOsm.
    5. Add reduced L-glutathione as a powder and swirl the container to dissolve it.
    6. Add hemin chloride from a freshly-prepared 4 mM stock (see Recipes).
    7. Adjust the pH to 6.5 by adding 5 N NaOH.
    8. Prepare the freezing point osmometer by turning it on for 30 min prior to measurements and then calibrate it with 100, 290, and 1,000 mOsm standards.
    9. Determine the osmolality of the CeM1 medium with the freezing point osmometer. The osmolality should be below the desired osmolality of 390 mOsm.
    10. Adjust the osmolality of the CeM1 medium to 390 mOsm/kg by adding additional trehalose (dissolved by swirling).
    11. Sterile filter the CeM1 medium through 0.22 µm 150 or 500 ml filter units.
    12. Transfer the sterile CeM1 medium to 15 ml or 50 ml polypropylene tubes so that it can be stored in aliquots with little air in the container (to limit evaporation and condensation). Store the aliquot tubes at 4 °C, wrapped in aluminum foil to protect them from light. The aliquots can be used for at least two months.

  3. Preparation of heat-killed OP50 bacteria
    1. Grow one liter of OP50 bacteria in 2x YT bacterial medium overnight at 37 °C.
    2. Spin down the bacteria at 3,700 x g for 30 min at 4 °C, decant the liquid.
    3. Resuspend in 5 ml of 0.9% NaCl and place in a 15 ml polypropylene tube.
    4. Incubate the tube in a water bath set at 62-67 °C for 24 h.
    5. Store heat-killed bacteria at 4 °C.
    Note: Do not spin down the heat-killed bacteria, which would compact the bacteria. Prior to using the bacteria, resuspend the solution by pipetting up and down with a 1,000 µl filter tip (after washing the shaft of the pipetter with 70% ethanol to sterilize it).

  4. Preparation of tumorous mutants
    1. A source of eggs for the procedure can be a mixed-stage population of tumorous germline mutants that contains gravid adults (i.e., adults with eggs in their body) and laid eggs. One 10 cm 3x NGM agar plate seeded with OP50 bacteria (see Recipes) and a somewhat dense population of mixed-stage animals will generally be adequate to provide sufficient eggs for the procedure. If using a temperature sensitive (ts) tumorous germline mutant, the 3x NGM agar plate should be incubated at the permissive temperature of 15-16 °C in a low-temperature incubator to allow the mutants to produce eggs. Several hundred eggs is sufficient for most small-scale experiments, e.g., 25 adults (derived from eggs) is used for isolation of germ cells for every well of a 24-well plate (each with 0.5 ml of medium).
    2. Collect C. elegans animals and eggs from the plate by rinsing with M9 buffer and scraping the eggs in M9 buffer with a cell scraper. The scraping is carried out by tilting the plate with one hand at an approximate angle of 30°, which makes the M9 buffer settle to the bottom part of the tilted plate. The cell scraper then is used with the other hand to move the cells and bacteria into the M9 liquid. The plate is then rotated so that regions with intact bacteria are brought to the top of the plate where they can be scraped into the liquid. When all the bacteria and eggs are in the M9 buffer, the liquid is transferred with a sterile glass Pasteur pipet to a 15 ml polystyrene tube.
    3. Spin down the animals and eggs by centrifugation at 930 x g for 1 min.
    4. Aspirate the liquid, and add 8 ml of sodium hypochlorite solution (see Recipes) and incubate with frequent inversions for approximately 3.5-4.5 min. During the incubation, occasionally place the 15 ml tube on a stereomicroscope to observe the animals. The incubation with sodium hypochlorite should be continued until almost all of the adults have broken in half, which releases their eggs.
    5. Spin down the eggs at 930 x g for 1 min, aspirate solution.
    6. Wash four times with sterile M9 solution (each time filling up the 15 ml tube).
    7. Use a sterile Pasteur pipet to place the sterile eggs (in a small volume of M9) onto an OP50-seeded 3x NGM plate. Although not required, if a more synchronized population of adults is desired, the sterile eggs can instead be placed in 15 ml of M9 with 5 µg/ml cholesterol in a 10 cm Petri dish, and left overnight at room temperature so that all of the eggs hatch and the resulting larvae arrest at the L1 larval stage. The L1 larvae can then be collected in a polystyrene 15 ml tube using a glass Pasteur pipet, spun down, excess liquid aspirated, and the eggs in a small volume of M9 (~100 µl) added to an OP50-seeded 3x NGM plate.
    8. Place the 3x NGM plate with eggs at 25 °C in a low-temperature incubator for four days. During this time period, the animals will develop to become adults with germline tumors (Figure 1).


      Figure 1. Stereomicroscope image of wild-type and tumorous mutant C. elegans adult hermaphrodites grown at 25 °C. Note that in the daf-16(mu86); cki-2(ok2105); glp-1(ar202) mutant, germline tumors are visible as white areas in the body (arrows). Scale bar = 100 µm.

    9. Collect the germline tumorous mutant animals from the plate by adding M9 solution to the edge of the plate and transfer the floating animals off the plate with a sterile Pasteur pipet into a 15 ml polystyrene tube (without disturbing the bacterial lawn).
    10. Wash the collected mutant animals four times with ~14 ml of M9 buffer to remove live bacteria (spinning at 230 x g for 1 min for each wash).
    11. Add the washed animals to a 12.5 cm2 cell culture flask that contains 2.5 ml of antibiotic-enriched PBS with heat-killed bacteria (see Recipes).
    12. Close the flask tightly and incubate at 25 °C overnight in a low-temperature incubator.

      Notes:

      1. If eggs fail to spin down in the sodium hypochlorite solution, then the density of the Bleach/sodium hypochlorite used in preparing the sodium hypochlorite solution is too high. In this case, after the initial incubation that dissolves the animals, add sterile water to fill the 15 ml tube and invert to mix prior to spinning down the eggs. This reduces the density of the solution so that the eggs will pellet.
      2. Occasionally, animals that will be used for germ cell isolation (prior to harvesting into antibiotic-containing PBS) are contaminated with fungus or bacteria other than OP50 (other bacteria generally appear either as denser bacterial colonies on the relatively thin OP50 bacterial lawn and/or as bacterial growths with altered color or texture). In this situation, we recommend aborting the experiment if long-term cultures are required, as continuing will generally lead to bacterial or fungal contamination in the in vitro culture. The contamination will generally not be overtly visible until the next day, and so isolated germ cells could be used for experiments within several hours of isolation.
      3. Polystyrene tubes are used to collect C. elegans larvae, adults, and eggs, because the animals do not stick to polystyrene. In contrast, if polypropylene tubes are used, animals and eggs will be lost during washes due to their sticking to the sides of the tube.
      4. Glass Pasteur pipets are used for transfers because eggs and animals do not stick to glass. In contrast, eggs and animals will stick to plastic pipet tips, so these should not be used.
      5. SARSTEDT cell scrapers are packaged as sterile and disposable, but they do not need to be sterile if they are used for collecting eggs prior to sodium hypochlorite treatment. In this situation, they can be reused after washing with soap.
      6. When scraping eggs from the plates, care should be taken to avoid scraping the edge of the plate. The sharp edge of the scraper hitting the elevated agar at the edge of the plate can chip off pieces of agar that would then be included with the eggs.
      7. The tumorous mutants are grown at 25 °C to activate the glp-1(ar202) ts allele. If tumorous germline mutant alleles are used that are not ts, then the animals can be grown at lower temperatures (e.g., 20 °C).

  5. Germ cell isolation
    1. Collect the animals from the antibiotic incubation into a 15 ml polystyrene tube and spin down (all spins with adult animals are 230 x g for 1 min).
    2. Wash the animals four times with sterile PBS (filling the tube).
    3. Wash one time with 5 ml of CeM1 medium.
    4. In a tissue culture hood, resuspend the washed animals in 2 ml of CeM1 medium and place in a 35 mm tissue culture dish.
    5. Prepare a second 35 mm tissue culture dish with a 120 µl circle of CeM1 medium in the center of the dish, which will be used for the isolation of germ cells.
    6. Take the plates out of the tissue culture hood to a bottom-illuminated stereomicroscope. While observing the animals through the stereomicroscope, transfer live animals (which move on their own or when prodded) using a platinum-wire worm pick from the 2 ml plate to the 120 µl circle of CeM1 medium in the second plate. Move the desired number of animals to the 120 µl circle (generally, 25 adult hermaphrodites are used per well of a 24-well plate, or 8 per well of a 96-well plate; we have harvested germ cells from up to 240 animals per one 120 µl circle). The worm pick for the transfer is a straightened platinum-wire with a flattened end, with a slight bend prior to the end so that when the wire is placed below multiple worms and lifted straight up, the worms are draped over both sides of the wire as it lifts out of the medium. The worms on the pick are then transferred to the media circle by placing the worms in the media and then dislodging them with a slight back and forth motion. Any non-moving worms that were transferred are removed with the flattened end of the wire pick.
    7. While visualizing through the stereomicroscope, cut the animals in the 120 µl circle into quarters using 21 gauge needles. To cut animals, one needle is placed on one side of an animal and the other needle is placed on the other side of the animal and then slid through the animal (next to the first needle), producing a scissor-like cutting action (Figure 2). Intact gonads that become extruded from animals are also cut.


      Figure 2. Dissection of animals to release germ cells. To dissect animals, needles are placed on either side of an animal as shown in (A). The needle in the background is brought forward next to the forward needle (arrow direction in A). The scissor-like action results in the dissection of the animal (B). The halves of the animal are then each dissected once more to produce smaller body pieces (as shown around the main animal).

    8. Bring the plate back to the tissue culture hood. Add 1 ml of CeM1 medium to the plate with a 1,000 µl pipet tip, while holding the plate at ~30° angle. Pick up the 1 ml of CeM1 with the pipet and eject it forcefully into the middle of the plate (where the plastic has been scored/scratched by the needles); repeat this a total of 20 times. Do not release all the medium from the pipet when ejecting, as this will generate bubbles that can kill cells due to the surface tension on the bubbles.
    9. Transfer all the liquid from the plate to the bottom of a 15 ml polypropylene tube.
    10. Pipet a second 1 ml of CeM1 medium eight times against the center of the plate (still held at ~30° angle), and add it to the same 15 ml tube.
    11. Add a third 1 ml of CeM1 medium directly to the 15 ml tube, and pipet the liquid up and down ~10 times (again avoiding pipetting all the liquid out of the pipet tip to prevent the formation of bubbles).
    12. Spin the tube at 21-230 x g for 1 min to pellet body parts and large cell aggregates. The choice of centrifuge speed affects the distribution of germ cells isolated: spinning at 21 x g will retain large cell aggregates in the supernatant; spinning at 230 x g will remove large cell aggregates, leaving only individual cells and small cell aggregates in the supernatant.
    13. Transfer the supernatant to a new 15 ml polypropylene tube and spin for 930 x g for 5 min to pellet the cells.
    14. Aspirate the supernatant, and resuspend the cell pellet in fresh CeM1 medium by gentling pipeting up and down with a 1,000 µl pipet tip that has a relatively wide opening (as found on the specified Corning pipet tips). The cells are transferred to tissue culture dish(es) based on the number of animals harvested. Typically, 0.08 ml is used for each well of a 96-well plate (with 8 animals/well); 0.5 ml for each well of a 24-well plate (with 25 animals/well), and 2 ml for culturing in a 35 mm tissue culture dish (with > 50 animals/dish). The ratio of cells to medium can be changed based on the experiment.
    15. If multi-well tissue culture dishes are used and the outside wells do not contain cells, then fill the outside wells with sterile PBS to limit evaporation from wells that contain isolated germ cells.
    16. Seal the edge of the tissue culture dish or plate with Parafilm (to limit evaporation).
    17. Incubate the plates at 25 °C in a low-temperature incubator in an airtight container.
    18. Viable germ cells can be maintained for periods of up to four weeks.
    19. Visualize isolated germ cells with a phase-contrast inverted microscope (Figure 3).


      Figure 3. Phase-contrast and Hoechst 33342 DNA stain of isolated C. elegans germ cells. Cells were imaged one day post isolation. Scale bar = 10 µm.

Data analysis

The numbers of live germ cells that are isolated in vitro can be determined as described in Chaudhari et al., 2016. To count live cells, the cells are stained with the live-cell stain calcein-AM, the dead-cell stain ethidium homodimer, and the DNA stain Hoechst 33342. Calcein-AM can freely diffuse into live cells but is then converted by cellular esterases to a fluorescent form that is unable to cross intact plasma membranes and so remains within the cell. Ethidium homodimer stains dead cells that lack an intact plasma membrane. The cell permeable dye Hoechst 33342 allows the visualization of nuclear DNA. The procedure to count live and dead cells is as follows.

  1. Isolated germ cells are gently mixed in the tissue culture medium with a filtered 1,000 µl pipet tip.
  2. 40 µl of the cells is transferred to a 0.5 ml centrifuge tube with a new 1,000 µl pipet tip.
    Note: A 1,000 µl pipet tip is recommended because the opening of the tip is large enough to prevent the cells from being damaged while they are drawn into the tip.
  3.  The stock mixture of Hoechst 33342, calcein-AM, and ethidium homodimer (see Recipes) is diluted 1:10 in CeM1, and then 1 µl of the dilution is mixed with the 40 µl of cells. The final concentration is 5 µg/ml Hoechst 33342; 1 µM calcein-AM; and 0.1 µM ethidium homodimer.
  4.  The tube with the cells is wrapped in aluminum foil (to block light) and incubated on a nutator for 30 min at room temperature.
  5. A pipet tip for a 20 µl pipetter is sterilely cut ~5 mm from the tip with a razor blade (sterilized by washing with 70% ethanol) in order to make a larger opening, and is used to resuspend the stained cells, and then ~10 µl of the solution is loaded into a hemacytometer.
  6.  The hemacytometer is analyzed on an upright compound fluorescence microscope. Live cells stain with calcein-AM (green) and contain nuclei that stain with Hoechst 33342 (blue), but lack staining with ethidium homodimer (red). Dead cells stain with ethidium homodimer, but lack staining with calcein-AM.

If hermaphrodites with large tumors are selected for dissection, up to 6,000 live germ cells can be isolated per hermaphrodite if a low speed is used for the initial spin (to remove animal parts and larger cell aggregates) (Chaudhari et al., 2016). The number of isolated germ cells will be reduced if higher speeds are used for the initial spin, as this will remove more aggregated germ cells. Analysis of the cells isolated from wild-type animals (from which live germ cells cannot be isolated due to their syncytial nature) vs. ET507 tumorous germline mutants showed that over 99% of the isolated cells from ET507 are germ cells (Chaudhari et al., 2016).

Recipes

  1. 2xYT bacterial medium
    16 g tryptone
    10 g yeast extract
    5 g NaCl
    1 L distilled water
    Add ingredients to a 2 L Erlenmeyer flask, cover with aluminum foil, and autoclave for 30 min
  2. LB bacterial medium
    10 g tryptone
    5 g yeast extract
    10 g NaCl
    0.2 ml 5 N NaOH
    1,000 ml distilled water
    Dissolve components by mixing with a stir bar. Aliquot to ten 100 ml bottles and autoclave for 20 min
    Note: This recipe is the conventional recipe for LB bacterial medium, but it differs from the original LB medium (Bertani, 1951) in that it does not contain 0.1% glucose.
  3. 3x NGM agar plates seeded with OP50 bacteria
    1. 3 g NaCl
      7.5 g Bacto-peptone
      20 g agar
      1 ml 5 mg/ml cholesterol (dissolved in 100% ethanol)
      1 ml 1 M MgSO4
      1 ml 0.1 M CaCl2
      972 ml distilled water
      Add to a 2 L Erlenmeyer flask that is then covered with aluminum foil
    2. Separately prepare a 50 ml Erlenmeyer flask or other small glass container (covered with aluminum foil) and filled with: 25 ml 1 M KH2PO4
    3. Autoclave the 2 L flask, small container, and a 1 L plastic pouring measuring cup with handle (covered with aluminum foil) for 30 min
    4. After autoclaving, add the 25 ml 1 M KH2PO4 to the 1 L in the flask. Swirl, pour into the 1 L measuring cup. Wait for the bubbles to disappear. Pour into 10 cm Petri dishes. Allow the agar plates to air dry for two days
    5. Grow an overnight culture of OP50 bacteria in LB medium. Put a small amount of the overnight OP50/LB culture onto each plate with a 10 ml glass pipet (that has a completely smooth tip), spread the bacteria on the surface of the plate with the glass pipet, leaving an ‘unseeded’ space around the edge of the plate
    6. Incubate the plates at room temperature for two days and then place in the plastic sleeves that originally held the Petri dishes, seal with tape, and store at 4 °C until needed. These plates can be used for one to two months

      Note: This recipe is a modification of the conventional NGM recipe (Sulston and Hodgkin, 1988); it differs in that it contains a three-fold higher concentration of peptone to allow thicker growth of bacteria.

  4. M9 buffer
    11.32 g Na2HPO4·7H2O
    3 g KH2PO4
    5 g NaCl
    0.012 g MgSO4
    Add distilled H2O to 1 L
    Aliquot to ten 100 ml bottles and autoclave for 20 min
    Note: This recipe is a modification of the conventional M9 buffer recipe (Sulston and Hodgkin, 1988); it differs in that it contains a lower concentration of MgSO4 to prevent salt precipitation after autoclaving.
  5. Sodium hypochlorite solution
    5.4 parts distilled water
    3 parts sodium hypochlorite (6%) or regular Bleach
    1.6 parts 5 N NaOH
    Prepare fresh in 15 ml or 50 ml polypropylene tube prior to use
  6. Creating a platinum-wire worm pick
    1. Cut a 3-4 cm piece of 0.25 mm-diameter platinum wire
    2. Wrap a long Pasteur pipet in a paper towel and break the glass at the point where the tapering starts
    3. Heat the broken end of the Pasteur pipet in a Bunsen burner so that it is completely melted. While holding the platinum wire with forceps or pliers, embed the platinum wire in the melted end of the Pasteur pipet (generally while both are in the Bunsen burner flame)
    4. Remove from the flame and allow the glass to cool. The end of the platinum wire can be flattened and shaped as desired
  7. Tetracycline stock
    1. Dissolve 12.5 mg/ml tetracycline in 70% ethanol and 30% distilled water
    2. Filter with a 150 ml or 500 ml PES-membrane filter unit with a separate bottom container
    3. Wrap the bottom filter container with aluminum foil to protect the tetracycline solution from light. Store at -20 °C for over six months
  8. Chloramphenicol stock
    Dissolve 34 mg/ml chloramphenicol in 100% ethanol
    Store at -20 °C for over six months
  9. Kanamycin stock
    Dissolve 25 mg/ml kanamycin in distilled water
    Filter sterilize and store at -20 °C for over six months
  10. Cholesterol stock
    Dissolve 10 mg/ml cholesterol in 100% ethanol
    Store at room temperature for over twelve months
  11. Antibiotic-enriched PBS with heat-killed bacteria
    Sterile PBS supplemented with:
    2% penicillin/streptomycin (from 100x stock)
    25 µg/ml tetracycline (from 12.5 mg/ml stock)
    34 µg/ml chloramphenicol (from 34 mg/ml stock)
    50 µg/ml kanamycin (from 50 mg/ml stock)
    0.2% normocin (from 500x stock)
    10 µg/ml cholesterol (from 10 mg/ml stock)
    Heat-killed bacteria are added to make the solution a lightly-cloudy white color (~50-200 µl of the heat-killed bacteria/2.5 ml PBS solution)
  12. Hemin chloride stock
    Prepare a fresh solution of 2 mM hemin chloride by dissolving 13 mg of hemin chloride in 9.8 ml water plus 0.2 ml 5 N NaOH. Prepare fresh each day
  13. Stock of Hoechst 33342, calcein-AM, and ethidium homodimer
    1. Prepare a 1.6 mM stock of ethidium homodimer in DMSO. Shield from light and store at -80 °C
    2. Prepare a 10 mg/ml stock of Hoechst 33342 in distilled water. Shield from light and store at -80 °C
    3. Prepare a 1.6 mM stock of calcein-AM in distilled water. Shield from light and store at -80 °C
    4. To create 40 µl of stock solution, mix 1 µl ethidium homodimer stock solution (40 µg/ml final concentration); 8 µl Hoechst 33342 stock solution (2 mg/ml final concentration); 10 µl calcein-AM stock solution (0.4 mg/ml final concentration); 3 µl DMSO; and 18 µl distilled water. The stock solution can be stored shielded from light at -20 °C or -80 °C

      Note: If one or more stain is too faint or bright, the concentration can be adjusted in the stock solution, with concomitant decreases or increases in the stain’s solvent (DMSO or water).

Acknowledgments

This protocol has been adapted from Chaudhari et al., 2016. Some C. elegans strains used to create strain ET507 were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work was supported by grants from NIH/NIGMS (R01GM074212) and NSF (MCB-1138454) to E.T.K.

References

  1. Bertani, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62(3): 293-300.
  2. Chaudhari, S. N., Mukherjee, M., Vagasi, A. S., Bi, G., Rahman, M. M., Nguyen, C. Q., Paul, L., Selhub, J. and Kipreos, E. T. (2016). Bacterial folates provide an exogenous signal for C. elegans germline stem cell proliferation. Dev Cell 38(1): 33-46.
  3. Hansen, D. and Schedl, T. (2013). Stem cell proliferation versus meiotic fate decision in Caenorhabditis elegans. Adv Exp Med Biol 757: 71-99.
  4. Kimble, J. and Seidel, H. (2013). C. elegans germline stem cells and their niche. StemBook. Harvard Stem Cell Institute. 1-12.
  5. Strange, K., Christensen, M. and Morrison, R. (2007). Primary culture of Caenorhabditis elegans developing embryo cells for electrophysiological, cell biological and molecular studies. Nat Protoc 2(4): 1003-1012.
  6. Sulston, J. and Hodgkin, J. (1988). The Nematode Caenorhabditis elegans. In: Wood, W. B. (Ed). Cold Spring Harbor Laboratory 587-606.
  7. Zhang, S. and Kuhn, J. R. (2013). Cell isolation and culture. WormBook 1-39.

简介

秀丽隐杆线虫种系是胚芽干细胞研究的重要模型系统。 野生型C。 线虫生殖细胞是合胞体,因此不能在体外培养中分离。 相比之下,来自肿瘤突变体的生殖细胞可以完全被细胞化并从突变动物中完整分离。 在这里,我们描述了从肿瘤突变体中分离生殖细胞的详细方案,其允许生殖细胞在体外原代培养中长时间维持。 该协议已从2016年Chaudhari等人改编。
【背景】秀丽生殖细胞在位于两个性腺臂的远端区域的两个成体干细胞龛中产生(Hansen和Schedl,2013; Kimble和Seidel,2013)。在野生型雌雄同体中,有丝分裂生殖细胞仅限于性腺臂的远端,干细胞生态位。野生型生殖细胞是合胞体,并且包含延伸通过性腺臂中心区域的共同细胞质的开口。 ℃。线虫肿瘤种系突变体在整个性腺中增加了生殖细胞的有丝分裂增殖。我们发现肿瘤种系突变体通常具有完整的细胞生殖细胞,其含有完整的质膜(Chaudhari等人,2016)。这种细胞化允许生殖细胞的分离及其在培养中的维持。该方案描述了分离和维持培养物中肿瘤突变体的生殖细胞的方法学和组织培养基。虽然已经描述了用于C的主要培养物的培养基。 elegans 胚胎和幼虫细胞(Strange等人,2007; Zhang和Kuhn,2013),生殖细胞在这种培养基中不能存活(Chaudhari等人。 ,2016)。我们为“C”制造了一种称为CeM1的生殖细胞培养基。 elegans medium 1'。我们预计媒介的其他迭代可以被给予后续的数字,例如,,'CeM2'。该方案首先在Chaudhari等人发表于2016年,允许在原代培养物中分离基本上纯净的生殖细胞群体及其维持。这种培养系统可以允许新的实验方法来探测生殖细胞生物学。线虫

关键字:秀丽隐杆线虫, 原代培养, 生殖干细胞, 组织培养

材料和试剂

  1. 聚苯乙烯管,15ml(Corning,Falcon ®,目录号:352099)
  2. 聚丙烯管,15毫升(康宁,目录号:25319-15)
  3. 聚丙烯管,50ml(Corning,目录号:25330-50)
  4. 过滤器吸头,100-1,000μl(Corning,目录号:4809)
  5. 过滤器吸头,20-200μl(Fisher Scientific,目录号:02-707-430)
  6. 过滤移液管头,2-20μl(Fisher Scientific,目录号:02-707-435)
  7. 过滤移液管头,0.1-10μl(Fisher Scientific,目录号:02-707-439)
  8. 过滤单元150 ml,PES 0.22μm(EMD Millipore,目录号:SCGPU01RE)
  9. 过滤单元500ml,PES0.22μm(EMD Millipore,目录号:SCGPU05RE)
  10. 铝箔
  11. 组织培养皿,35 x 10mm(Corning,Falcon ®,目录号:353001)
  12. 组织培养皿,24孔(康宁,目录号:3524)
  13. 组织培养皿96孔(康宁,目录编号:3596)
  14. 细胞刮板,39厘米,一次性(SARSTEDT,目录号:83.1831)
  15. 血清学移液管,一次性10ml(Fisher Scientific,目录号:13-678-11E)
  16. 组织培养瓶12.5cm 2(Corning,Falcon ,目录号:353018)
  17. 铂金线,0.25mm(Alfa Aesar,目录号:10288)
  18. 针,21 G x 1½(BD,目录号:305167)
  19. 石蜡膜
  20. 0.5ml离心管
  21. 微量离心管,1.5 ml(Fisher Scientific,目录号:05-408-129)
  22. 剃刀刀片,单刃
  23. 纸巾
  24. ℃。线虫肿瘤种系突变株ET507,daf-16(mu86)I; cki-2(ok2105) II; glp-1(ar202) III
  25. 大肠杆菌菌株OP50
    注意:
    1. 我们没有进行测试,以确定其他制造商的材料和试剂如何在生殖细胞分离和培养中起作用,除了使用STARSTEDT 96孔板(非组织培养处理)(STARSTEDT,目录号:82.1581.001)用于体外培养生殖细胞,导致生殖细胞过早死亡。
    2. ℃。线虫种系肿瘤突变株ET507(以及其他肿瘤突变菌株)和大肠杆菌细菌OP50可从Caenorhabditis遗传学中心(CGC)获得, http://www.cgc.cbs.umn.edu em>
  26. 胎牛血清(FBS)(Atlanta Biologicals,目录号:S11550)
  27. Amberlite IRA 400-CL(Sigma-Aldrich,目录号:247669)
  28. 炭葡聚糖(Sigma-Aldrich,目录号:C6241)
  29. 液氮
  30. 磷酸盐缓冲盐水(PBS)(GE Healthcare,HyClone TM,目录号:SH30256.01)
  31. 施耐德的昆虫培养基(Thermo Fisher Scientific,Gibco TM,目录号:21720024)
  32. Leibovitz的L-15无酚红培养基(Thermo Fisher Scientific,Gibco TM ,目录号:21083027)
  33. 青霉素/链霉素(Sigma-Aldrich,目录号:P4333)
  34. 血红素氯化物(MP Biomedicals,目录号:0219402501)
  35. RPMI维生素(Sigma-Aldrich,目录号:R7256)
  36. L-谷胱甘肽,还原(Sigma-Aldrich,目录号:G4251)
  37. Normocin(InvivoGen,目录号:ant-nr-1)
  38. 海藻糖(Sigma-Aldrich,目录号:T0167)
  39. 渗透压标准,100 mmol / kg(Wescor,目录号:OA-010)
  40. 重量渗透压标准,290mmol / kg(Wescor,目录号:OA-029)
  41. 渗透摩尔浓度标准,1,000 mmol / kg(Wescor,目录号:OA-100)
  42. 水,分子生物学级(GE Healthcare,HyClone TM,目录号:SH30538.02)
  43. 胰蛋白胨(Fisher Scientific,目录号:BP1421-500)
  44. 酵母提取物(Fisher Scientific,目录号:BP9727-2)
  45. 氯化钠(NaCl)(Avantor Performance Materials,J.T.Baker ,目录号:3624-05)
  46. 氢氧化钠(NaOH)(Avantor Performance Materials,J.T.Baker ,目录号:3728-01)
  47. 细菌蛋白胨(BD,Bacto TM,目录号:211677)
  48. 琼脂(RPI,目录号:A20020-5000)
  49. 胆固醇(Avantor Performance Materials,J.T.Baker ®,目录号:1580-01)
  50. 乙醇,100%(用蒸馏水制成70%)(Decon Labs,目录号:2716)
  51. 无水硫酸镁(MgSO 4)(Avantor Performance Materials,J.T.Baker ,目录号:2506-01)
  52. 氯化钙(CaCl 2)(Avantor Performance Materials,J.T.Baker ,目录号:1313-01)
  53. 磷酸二氢钾(KH 2 O 3 PO 4)(Avantor Performance Materials,JTBaker,目录号:3246-05) >
  54. 磷酸钠,七水合物(Na 2 HPO 4·7H 2 O)(Fisher Scientific,目录号:S373-3)
  55. 次氯酸钠,6%(RICCA Chemical,目录号:7495.7-32)
  56. 四环素(Sigma-Aldrich,目录号:87128)
  57. 氯霉素(RPI,目录号:C61000-25.0)
  58. 卡那霉素(Fisher Scientific,目录号:BP906-5)
  59. 乙锭同型二聚体(Biotium,目录号:40010)
  60. 二甲基亚砜(DMSO)(Fisher Scientific,目录号:D128-500)
  61. Hoechst 33342(Sigma-Aldrich,目录号:B2261)
  62. Calcein-AM(Biotium,目录号:80011)
  63. 2xYT细菌培养基(参见食谱)
  64. LB细菌培养基(见食谱)
  65. 3x NGM琼脂平板接种OP50细菌(见食谱)
  66. M9缓冲区(见配方)
  67. 次氯酸钠溶液(见配方)
  68. 铂金线虫(见配方)
  69. 四环素(见食谱)
  70. 氯霉素原料(见食谱)
  71. 卡那霉素原料(见食谱)
  72. 胆固醇(见配方)
  73. 具有热杀菌细菌的富含抗生素的PBS(参见食谱)
  74. 血红素库存(见配方)
  75. Hoechst 33342,钙黄绿素AM和同源二聚体(见配方)的库存

设备

  1. 温控水浴
  2. 台式离心机,带有15毫升和50毫升管的摆动式转子
  3. 旋转器,单速(Barnstead Thermolyne,目录号:C415110)
  4. 500毫升塑料瓶
  5. 凝固点渗透压计(Advanced Digimatic Osmometer 3DII,Advanced Instruments,型号:Model 3D2)
  6. 低温培养箱
  7. 底部照明立体显微镜与磨砂玻璃舞台或磨砂镜(各种尼康或徕卡型号)
  8. 气密容器(LockandLock,各种尺寸的锁和容器从Amazon获得)
  9. 相位反转复合显微镜(各种尼康,徕卡或具有10倍和20倍目标的奥林巴斯型号)
  10. Nutator(BD,目录号:421105)
  11. 血细胞计数器(Reichert Bright-Line,Hauser Scientific,目录号:1492)
  12. 直立荧光复合显微镜(各种尼康,徕卡或具有5x,20x,40x和64x目标的Olympus型号)
  13. 2升锥形瓶(Corning,PYREX ,目录号:4980-2L)
  14. 高压灭菌器
  15. 50ml锥形瓶(Corning,PYREX ,目录号:4980-50)
  16. 塑料1 L带手柄的量杯(Fisher Scientific,目录号:02-543-36C)
  17. 搅拌棒
  18. 瓶125毫升,玻璃(WHEATON,目录号:219815)
  19. 本生灯
  20. 分析结果
  21. pH计
  22. 移液器控制器(Pipet-Aid)(Drummond Scientific,目录号:4-000-110)
  23. 巴斯德移液器,9英寸玻璃(Fisher Scientific,目录号:22-063172)
  24. 巴斯德移液器灯泡(Fisher Scientific,目录号:03-448-21)
  25. 层流组织培养罩
  26. 吸气器

程序

  1. 预处理FBS
    1. 如果FBS以等分试样(-20℃或-80℃)冷冻,则解冻适量的FBS以制备所需体积的CeM1培养基(FBS的终浓度为8%)。注意,FBS的预处理由于在炭 - 葡聚糖中捕获FBS而导致体积的〜20%的损失,例如,25ml的FBS将产生约20ml的预处理的FBS。 FBS可以在室温或37°C水浴中解冻。预处理包含两个隔夜培育步骤。 FBS与Amberlite IRA 400-CL珠粒和木炭葡聚糖的孵育可除去疏水性分子,如类固醇激素。预处理步骤需要在CeM1培养基中维持生殖细胞活力(Chaudhari等人,2016)。不知道预防如何改变FBS以改善细胞存活。
    2. 通过在温度控制的水浴中,在56-65℃温育30分钟,使偶尔的旋转(每5-10分钟),将热解灭活。
    3. 预洗一定量的Amberlite IRA 400-CL珠粒,其基于将经受预处理的每ml FBS的最终浓度为50mg的珠粒;对于25ml的FBS,这将是1.25g珠。对于预洗,将珠子放在15ml或50ml聚丙烯管中。通过向管中加入分子级水(〜13或〜45ml,取决于是否使用15ml或50ml管)并将管反转多次,将珠预先洗涤。通过在台式离心机中以930×g离心1分钟收集珠粒。吸出珠子上方的水。重复洗两次。通过将1000μl移液管针头放在管底部并移出水分,清除珠子之间的残留水。
    4. 用洗涤的Amberlite IRA 400-CL珠将热灭活的FBS加入到管中
    5. 将FBS和Amberlite IRA 400-CL珠在室温下以单速旋转器旋转4-6小时。
    6. 预先洗涤第二组Amberlite IRA 400-CL珠(1.25g,25ml FBS),如步骤A3所示。
    7. 将珠子(930 x g)旋转1分钟,并将FBS转移到第二批洗涤的Amberlite IRA 400-CL珠粒中。
    8. 旋转FBS和珠子4°C旋转过夜。
    9. 第二天,准备木炭葡聚糖与FBS孵育。木炭葡聚糖的量基于每毫升FBS最终浓度为100mg的木炭葡聚糖;对于25ml FBS,这将是2.5g的木炭葡聚糖。将木炭葡聚糖预先压在称重盘的一侧,以分解成块。放入15ml或50ml聚丙烯管中
    10. 将Amberlite IRA 400-CL珠子(930 x g)旋转1分钟,然后将FBS转移到带有木炭葡聚糖的管中。
    11. 旋转FBS与木葡萄糖在4°C旋转器过夜。
    12. 通过两次连续离心除去木炭葡聚糖,每次离心分别为2,850 xg 30分钟,4℃,然后将FBS转移到新的聚丙烯管(留下一小部分,约100μl,超过颗粒,以确保没有留下来自颗粒的残留物)。
    13. 已经通过热灭活处理的FBS和与Amberlite IRA 400-CL和木炭葡聚糖的温育可以立即使用,在4℃下储存一至两周,或储存更长时间(几个月或几年) )在-80℃(快速冷冻等分液体在液氮中)

      注意:

      1. 不同批次的FBS对培养物中生殖细胞的长期存活有很大的影响。从FBS制造商/分销商那里获得多个批次的样品将有利于在购买特定批次的FBS之前测试生殖细胞存活。
      2. 在整个协议中,离心机速度基于具有摆动斗转子的Beckman Coulter Allegra X-15R台式离心机,其具有以下rpm至rcf(x g)转换:300rpm = 21×g; 1,000rpm = 230×g; 2,000rpm = 930×g; 3500 rpm = 2,850 x g; 4,000 rpm = 3,700 x g。

  2. 制备CeM1培养基
    1. 在没有用肥皂(例如,500ml以前保持水,PBS或组织培养基的塑料瓶)洗涤的瓶子中制备CeM1培养基,并通过用蒸馏水冲洗和空气干燥)。如果制备的培养基少于50ml,则可以用50ml的聚丙烯管制成。
    2. CeM1成分的百分比/浓度见表1.最初,添加Schneider的昆虫培养基,Leobovitz的无酚红的L-15培养基,预处理的FBS,青霉素/链霉素,RPMI维生素和诺卡诺霉素。

      表1. CeM1介质成分


    3. 加入10毫克/毫升乙醇中的胆固醇(参见食谱)。
    4. 加入18毫克/毫升海藻糖,旋转瓶子溶解粉末。这应产生低于所需390 mOsm的渗透压。
    5. 加入还原的L-谷胱甘肽作为粉末,旋转容器溶解
    6. 从新鲜制备的4mM储备液中加入氯化血红素(参见食谱)。
    7. 通过加入5N NaOH将pH调节至6.5
    8. 在测量之前将其打开30分钟,然后用100,290和1,000 mOsm标准进行校准,准备冰点渗透压计。
    9. 用凝固点渗透压测定CeM1培养基的重量摩尔渗透压浓度。重量摩尔渗透压浓度应低于390 mOsm所需的渗透压。
    10. 通过添加额外的海藻糖(通过旋转溶解)将CeM1培养基的重量克分子渗透浓度调节至390mOsm / kg。
    11. 通过0.22μm的150或500 ml过滤器单位对CeM1培养基进行无菌过滤
    12. 将无菌CeM1培养基转移到15ml或50ml聚丙烯管中,使其可以以少量空气等量存储在容器中(以限制蒸发和冷凝)。将等分试管存放在4℃,包裹在铝箔中以保护其免受光照。等分试样可以使用至少两个月。

  3. 制备热灭活的OP50细菌
    1. 在2×YT细菌培养基中培养1升OP50细菌在37℃下过夜
    2. 在4℃下将细菌以3,700 x g 旋转30分钟,倾倒液体。
    3. 重悬于5ml的0.9%NaCl中,置于15ml聚丙烯管中
    4. 将管子置于62-67℃的水浴中孵育24小时
    5. 在4℃下储存热灭菌细菌。

      注意:不要旋转热灭菌的细菌,这会压制细菌。在使用细菌之前,用1,000μl过滤嘴(用70%乙醇清洗移液器的轴进行消毒),上下移动,重新悬浮溶液。

  4. 肿瘤突变体的制备
    1. 该程序的卵源可以是混合种群的肿瘤种系突变体,其包含妊娠成虫(即,即,其身体中具有卵的成虫)和产卵。接种OP50细菌的一个10cm 3x NGM琼脂平板(参见食谱)和混合阶段动物的稍微密集的群体通常将足以提供足够的蛋用于该程序。如果使用温度敏感(ts)肿瘤种系突变体,3x NGM琼脂平板应在15-16℃的允许温度下在低温培养箱中孵育,以使突变体产生蛋。大多数小规模实验中有几百个蛋已经足够了,例如,25只成年人(来自鸡蛋)用于分离24孔板每个孔的生殖细胞(每个具有0.5ml中)。
    2. 收集C.线虫通过用M9缓冲液冲洗并用细胞刮刀在M9缓冲液中刮洗鸡蛋,从板中取出。刮削是通过一只手倾斜板以大约30°的角度进行的,这使得M9缓冲器沉降到倾斜板的底部。另一方面,使用细胞刮刀将细胞和细菌移动到M9液体中。然后将板旋转,使得具有完整细菌的区域被带到板的顶部,在那里它们可以被刮到液体中。当所有细菌和鸡蛋都在M9缓冲液中时,将液体用无菌玻璃巴斯德移液管转移到15ml聚苯乙烯管中。
    3. 通过在930 x g下离心1分钟来旋转动物和鸡蛋。
    4. 吸入液体,并加入8 ml次氯酸钠溶液(参见食谱),并频繁反转孵育约3.5-4.5分钟。在孵育期间,偶尔将15 ml管置于立体显微镜上观察动物。应该继续与次氯酸钠孵育,直到几乎所有的成年人都破碎了一半,释放出卵子。
    5. 将930 x x 的鸡蛋旋转1分钟,吸出溶液
    6. 用无菌M9溶液洗涤四次(每次充满15ml管)。
    7. 使用无菌巴斯德吸管将无菌卵(少量M9)置于OP50种子3x NGM板上。尽管不是必需的,如果需要更成熟的成年人群,则将无菌卵代替放置在10毫升培养皿中的5毫升/ 5升胆固醇的15毫升M9中,并在室温下放置过夜鸡蛋孵化,并在L1幼虫阶段捕获由此产生的幼虫。然后可以使用玻璃巴斯德移液管将L1幼虫收集在聚苯乙烯15ml管中,旋转下来,抽吸多余液体,并将小量的M9(〜100μl)的卵加入到OP50种子的3x NGM板中。
    8. 将3x NGM板在25℃的低温培养箱中放置鸡蛋4天。在此期间,动物将发育成为具有种系肿瘤的成年人(图1)

      图1.野生型和肿瘤突变体的立体显微镜图像 C。线虫 成年雌雄同株在25°C生长。 请注意,在daf-16(mu86)中; CKI-2(ok2105); glp-1(ar202)突变体,种系肿瘤在体内可见为白色区域(箭头)。比例尺= 100μm。

    9. 通过将M9溶液加入到板的边缘并从无菌的巴斯德移液器将浮动的动物从板上转移到15ml聚苯乙烯管中(不干扰细菌草坪),从板中收集种系肿瘤突变体动物。
    10. 用〜14ml M9缓冲液洗涤收集的突变体动物四次以除去活细菌(每次洗涤230℃旋转1分钟)。
    11. 将洗涤的动物加入到含有2.5ml含有热灭活细菌的富含抗生素的PBS的12.5cm 2细胞培养瓶中(参见食谱)。
    12. 紧紧关上烧瓶,并在低温培养箱中于25℃孵育过夜

      注意:

      1. 如果鸡蛋在次氯酸钠溶液中不能脱落,则用于制备次氯酸钠溶液的漂白剂/次氯酸钠的密度太高。在这种情况下,在溶解动物的初次孵育后,加入无菌水以填充15ml管,并在旋转鸡蛋之前反转混合。这样可以降低溶液的密度,使鸡蛋沉淀。
      2. 偶尔,将用于生殖细胞分离的动物(在收获含抗生素的PBS之前)被除了OP50之外的真菌或细菌污染(其他细菌通常在相对较薄的OP50细菌草坪上显示为更密集的细菌菌落和/或具有改变的颜色或纹理的细菌生长)。在这种情况下,如果需要长期培养,我们建议中止实验,因为一般会导致体外培养中的细菌或真菌污染。污染物一般不会在第二天才明显可见,因此分离的生殖细胞可以在隔离的几个小时内用于实验。
      3. 聚苯乙烯管用于收集线虫幼虫,成虫和鸡蛋,因为动物不粘在聚苯乙烯上。相比之下,如果使用聚丙烯管,则由于它们粘在管的两侧而在洗涤期间将会丢失动物和鸡蛋。
      4. 玻璃巴斯德移液器用于转运,因为鸡蛋和动物不粘玻璃。相比之下,鸡蛋和动物会坚持使用塑料吸头,因此不应该使用。
      5. SARSTEDT细胞刮刀包装为无菌和一次性,但如果在次氯酸钠处理之前用于收集蛋,则它们不需要无菌。在这种情况下,可以用肥皂清洗后重复使用。
      6. 从板上刮鸡蛋时,要注意避免刮板的边缘。刮刀的尖锐边缘撞击板上边缘的高琼脂,可以剥下随之而来的琼脂片。
      7. 肿瘤突变体在25℃生长以激活glp-1(ar202)ts等位基因。如果使用不是ts的肿瘤种系突变等位基因,则可以在较低温度(例如20℃)下生长动物。

  5. 生殖细胞分离
    1. 将动物从抗生素培养物中收集到15ml聚苯乙烯管中并旋转(所有与成年动物的旋转物230μg×1分钟)。
    2. 用无菌PBS(灌装管)清洗动物四次。
    3. 用5ml CeM1培养基洗一次。
    4. 在组织培养罩中,将洗涤的动物重悬于2ml CeM1培养基中并置于35mm组织培养皿中。
    5. 准备第二个35毫米的组织培养皿,在培养皿中心用120微升的CeM1培养基圈,将其用于分离生殖细胞。
    6. 将板从组织培养罩中取出到底部照明的立体显微镜。在通过立体显微镜观察动物时,使用铂线蠕虫从2ml板转移到活动动物(其自身移动或移动)到第二盘中的120μl圆形的CeM1培养基。将所需数量的动物移动到120μl的圆圈(通常每个孔用24孔板,每个孔使用25个成年雌雄同株,或96孔板每孔8个;我们收获的生殖细胞最多可达240只动物一个120μl圆)。用于转移的蜗杆皮是带有扁平端的拉直的铂线,在端部之前略微弯曲,使得当线被放置在多个蜗杆下方并且被直线抬起时,蜗杆被覆盖在线的两侧因为它从媒介中提升出来。然后,通过将蠕虫放在介质中,然后用轻微的来回运动将它们移动到介质圈。移动的任何不动的蠕虫都会用导线的扁平端移除。
    7. 在通过立体显微镜观察时,使用21号针将120μl圆圈中的动物切成四分之一。为了切割动物,将一只针放置在动物的一侧,将另一根针放置在动物的另一侧,然后滑动通过动物(第一针的第二针),产生剪刀状切割动作(图2)。从动物挤出的完整的性腺也被切断。


      图2.动物释放生殖细胞的解剖为了解剖动物,将针放置在动物的任一侧,如(A)所示。背景中的针沿前针(A中的箭头方向)前进。剪刀式动作导致动物的解剖(B)。然后每次解剖动物的一半以产生较小的身体部位(如主动物周围所示)。

    8. 将板带回组织培养罩。将1ml CeM1培养基加入到具有1000μl移液管末端的板中,同时将板保持在〜30°角。用移液管取出1 ml的CeM1,并将其强力推出到板的中间(塑料已被针刻划/划伤);共重复20次。在喷射时不要从吸管中释放出所有的介质,因为这会产生气泡,因为气泡表面张力会导致细胞死亡。
    9. 将所有液体从板上转移到15ml聚丙烯管的底部。
    10. 将第二个1ml的CeM1培养基相对于板的中心(仍然保持在-30°角)八次,并加入到相同的15ml管中。
    11. 将第三个1ml CeM1培养基直接加入到15ml管中,并将液体上下移动〜10次(再次避免将移液管中的所有液体移出吸头,以防止形成气泡)。
    12. 将管子以21-230×g旋转1分钟以沉淀身体部位和大细胞聚集体。离心速度的选择影响分离的生殖细胞的分布:在21×g下旋转将在上清液中保留大的细胞聚集体;在230×g下旋转将去除大的细胞聚集体,在上清液中仅留下单个细胞和小细胞聚集体。
    13. 将上清液转移到新的15ml聚丙烯管中,并旋转930×g 5分钟以沉淀细胞。
    14. 吸出上清液,并通过用具有相对较宽的开口的1,000μl移液管吸头(通过指定的康宁移液器吸头)发现,用新鲜的CeM1培养基重悬细胞沉淀。基于收获的动物数将细胞转移到组织培养皿中。通常,对于96孔板(8只动物/孔)的每个孔使用0.08ml。对于24孔板(25只动物/孔)的每个孔0.5ml,和2ml用于在35mm组织培养皿(> 50个动物/皿)中培养。可以根据实验改变细胞与培养基的比例。
    15. 如果使用多孔组织培养皿并且外部孔不含细胞,则用无菌PBS填充外部孔以限制从包含分离的生殖细胞的孔中蒸发。
    16. 用Parafilm密封组织培养皿或板的边缘(以限制蒸发)。
    17. 在25℃的温度下,在密闭容器中的低温培养箱中孵育
    18. 可行的生殖细胞可以维持长达四周。
    19. 用相差倒置显微镜观察分离的生殖细胞(图3)

      图3.分离的相对比和Hoechst 33342 DNA染色 C。线虫 生殖细胞。细胞在分离后一天成像。比例尺= 10μm

数据分析

可以按照Chaudhari等人,2016所述确定体外分离的活的生殖细胞的数量。为了计数活细胞,细胞用活的细胞染色钙黄质素-AM,死细胞染色乙锭同型二聚体和DNA染色剂Hoechst 33342.Calcein-AM可以自由地扩散到活细胞中,然后被细胞酯酶转化成不能穿过完整质膜的荧光形式并因此保留在细胞内。乙二胺二聚体污染缺乏完整质膜的死细胞。细胞渗透染料Hoechst 33342允许核DNA的可视化。计数活细胞和死细胞的程序如下。

  1. 将分离的生殖细胞在组织培养基中用过滤的1,000μl移液管末端轻轻混合。
  2. 将40μl的细胞转移到0.5ml离心管中,加入新的1,000μl移液管。
    注意:建议使用1,000μl的吸头,因为尖端的开口足够大,以防止细胞在吸入尖端时被损坏。
  3. 将Hoechst 33342,钙黄绿素AM和乙锭同型二聚体(见食谱)的混合物在CeM1中以1:10稀释,然后将1μl稀释液与40μl细胞混合。最终浓度为5μg/ ml Hoechst 33342; 1μM钙黄绿素AM;和0.1μM乙锭同二聚体。
  4. 将细胞管包裹在铝箔(阻挡光)中,并在室温下在养分器上孵育30分钟。
  5. 将20μl移液管的移液管末端用剃刀刀片(从70%乙醇洗涤消毒灭菌)至尖端约5毫米,以使开口更大,并用于重悬染染色细胞,然后〜将10μl溶液加载到血细胞计数器中。
  6. 在直立的化合物荧光显微镜上分析血细胞计数器。活细胞用钙黄绿素AM(绿色)染色,含有Hoechst 33342(蓝色)染色的细胞核,但缺乏用同型二聚体(红色)染色。死细胞用乙二胺二聚体染色,但缺乏钙黄绿素AM染色

如果与大肿瘤的雌雄同体被选择用于解剖,如果使用低速用于初始旋转(去除动物部分和较大的细胞聚集体),则雌雄同体可以分离出高达6,000个活的生殖细胞(Chaudhari等人, / em>,2016)。如果使用较高的速度进行初始旋转,则分离的生殖细胞的数量将会减少,因为这将消除更多的聚集的生殖细胞。从野生型动物(活细胞因其合胞体性质不能分离的细胞)分离的细胞对ET507肿瘤种系突变体的分析显示,来自ET507的分离的细胞中有超过99%是生殖细胞(Chaudhari < et al。,2016)。

食谱

  1. 2xYT细菌培养基 16克胰蛋白胨
    10g酵母提取物
    5克NaCl
    1升蒸馏水
    向2升锥形瓶中加入成分,盖上铝箔,高压灭菌30分钟
  2. LB细菌培养基
    10克胰蛋白胨
    5克酵母提取物
    10克NaCl
    0.2 ml 5 N NaOH 1000毫升蒸馏水
    通过与搅拌棒混合来溶解组分。等分至十个100ml瓶子,高压灭菌20分钟 注意:该配方是LB细菌培养基的常规配方,但与原始LB培养基(Bertani,1951)不同之处在于不含0.1%葡萄糖。
  3. 3x NGM琼脂平板接种OP50细菌
    1. 3克NaCl
      7.5g细菌蛋白胨
      20克琼脂
      1 ml 5 mg / ml胆固醇(溶于100%乙醇中)
      1ml 1M MgSO 4
      1ml 0.1M CaCl 2
      972毫升蒸馏水
      加入2升三角烧瓶中,然后用铝箔覆盖
    2. 分别制备50ml锥形瓶或其他小玻璃容器(用铝箔覆盖),并装满:25ml 1M KH 2 PO 4 /
    3. 将2L烧瓶,小容器和1L带手柄(铝箔覆盖)的塑料浇注量杯高压灭菌30分钟
    4. 高压灭菌后,加入烧瓶中的1升25毫升1M KH 2 O 3 PO 4。旋转,倒入1升量杯中。等待气泡消失。倒入10厘米培养皿。让琼脂板空气干燥两天
    5. 在LB培养基中生长OP50细菌的过夜培养物。将少量的过夜的OP50 / LB培养物用10毫升的玻璃移液管(具有完全光滑的尖端)在每个平板上,用玻璃移液管将细菌铺展在板的表面上,留下一个“未固化”的空间板的边缘
    6. 将板在室温下孵育两天,然后放在原来保持培养皿的塑料套筒中,用胶带密封,并在4℃下储存直到需要。这些板可以使用一到两个月

      注意:该配方是常规NGM配方的修改(Sulston和Hodgkin,1988);它的不同之处在于它含有三倍高浓度的蛋白胨,以允许细菌生长较为繁琐

  4. M9缓冲区
    11.32g Na 2 HPO 4·7H 2 O
    3g KH 2 PO 4
    5克NaCl
    0.012g MgSO 4
    将蒸馏的H 2 O加入1L
    等分至十个100ml瓶子,高压灭菌20分钟 注意:该配方是常规M9缓冲配方的修改(Sulston和Hodgkin,1988);它的不同之处在于它含有较低浓度的MgSO 4,以防止高压灭菌后的盐沉淀。
  5. 次氯酸钠溶液
    5.4份蒸馏水
    3份次氯酸钠(6%)或常规漂白剂
    1.6份5N NaOH
    在使用前准备新鲜的15 ml或50 ml聚丙烯管
  6. 创建一个铂金线虫
    1. 切割3-4厘米的0.25毫米直径的铂丝线,
    2. 将一个长的巴斯德吸管裹在纸巾上,并在锥形开始点的位置打破玻璃
    3. 在本生灯泡中加热巴斯德吸管的破碎端,使其完全熔化。在用镊子或钳子夹住铂金线的同时,将铂丝线插入巴斯德吸管的熔化端(通常两者都在本生灯火焰中)
    4. 从火焰中取出并让玻璃冷却。铂线的末端可以根据需要变扁和成型
  7. 四环素原料
    1. 将12.5 mg / ml四环素溶于70%乙醇和30%蒸馏水中
    2. 用150 ml或500 ml PES-膜过滤器过滤,并带有独立的底部容器
    3. 用铝箔包装底部过滤器容器,以保护四环素溶液免受光照。在-20°C储存六个月以上
  8. 氯霉素股票
    将34 mg / ml氯霉素溶于100%乙醇中 在-20°C储存六个月以上
  9. 卡那霉素库存
    将25 mg / ml卡那霉素溶于蒸馏水中 过滤灭菌并在-20°C储存六个月以上
  10. 胆固醇库存
    将10 mg / ml胆固醇溶于100%乙醇中 在室温下储存超过十二个月
  11. 具有热杀菌细菌的富含抗生素的PBS
    无菌PBS补充:
    2%青霉素/链霉素(100x原料)
    25μg/ ml四环素(12.5mg / ml储备)
    34μg/ ml氯霉素(34mg / ml储备)
    50μg/ ml卡那霉素(50mg / ml储备)
    0.2%normocin(来自500x股票)
    10μg/ ml胆固醇(来自10mg / ml原料)
    添加热灭菌的细菌使溶液呈淡白色(约50-200μl热杀菌细菌/ 2.5ml PBS溶液)
  12. 血红素氯化物库存
    通过将13mg海明氯溶解在9.8ml水加0.2ml 5N NaOH中来制备2mM氯化高锰酸盐的新鲜溶液。每天准备新鲜
  13. Hoechst 33342,钙黄绿素AM和同源二聚体乙炔的库存
    1. 在DMSO中制备1.6mM的同源二聚体的股份。从-80°C光线保存,然后存放在-80°C
    2. 在蒸馏水中制备10 mg / ml的Hoechst 33342储存液。从-80°C光线保存,然后存放在-80°C
    3. 在蒸馏水中制备1.6mM钙黄绿素AM的原料。从-80°C光线保存,然后存放在-80°C
    4. 为了产生40μl储备溶液,混合1μl乙二胺二聚体储备溶液(终浓度为40μg/ ml); 8μlHoechst 33342储备溶液(2mg / ml终浓度); 10μl钙黄绿素AM储备液(0.4mg / ml终浓度); 3μlDMSO;和18μl蒸馏水。储存液可以保存在-20°C或-80°C的光线下

      注意:如果一个或多个污渍太微弱或明亮,则可以在储备溶液中调节浓度,同时降低或增加污渍溶剂(DMSO或水)。

致谢

该协议已经从Chaudhari等人,2016年改编。一些C。用于产生菌株ET507的线虫株由CGC提供,由NIH研究基础设施办公室(P40 OD010440)资助。这项工作得到NIH / NIGMS(R01GM074212)和NSF(MCB-1138454)授予E.T.K.的授权。

参考

  1. Bertani,G.(1951)。关于溶菌发生的研究。 I.通过溶菌原大肠杆菌释放噬菌体的方式。 J Bacteriol 62(3):293-300。
  2. Chaudhari,SN,Mukherjee,M.,Vagasi,AS,Bi,G.,Rahman,MM,Nguyen,CQ,Paul,L.,Selhub,J.and Kipreos,ET(2016)。&lt; a class = ke-insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/27404357“target =”_ blank“>细菌叶酸提供了秀丽隐杆线虫的外源信号。细胞增殖。 Dev Cell 38(1):33-46。
  3. Hansen,D.和Schedl,T。(2013)。干细胞增殖与秀丽隐杆线虫的减数分裂命运决定。 Adv Exp Med Biol 757:71-99。
  4. Kimble,J.和Seidel,H。(2013)。&nbsp; ℃。线虫种系干细胞及其生态位。哈佛干细胞研究所。 1-12。
  5. Strange,K.,Christensen,M.and Morrison,R。(2007)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/17446899”目标=“_ blank”>秀丽隐杆线虫原代培养开发用于电生理,细胞生物学和分子研究的胚胎细胞。 Nat Protoc 2(4):1003- 1012.
  6. Sulston,J.和Hodgkin,J。(1988)。线虫秀丽隐杆线虫 在:Wood,WB(Ed)。冷泉港实验室 587-606。
  7. Zhang,S.and Kuhn,JR(2013)。&nbsp; 细胞分离和培养。 WormBook 1-39。
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引用:Vagasi, A. S., Rahman, M. M., Chaudhari, S. N. and Kipreos, E. T. (2017). Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants. Bio-protocol 7(15): e2424. DOI: 10.21769/BioProtoc.2424.
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