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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

AV Alexandra S. Vagasi
MR Mohammad M. Rahman
SC Snehal N. Chaudhari
EK Edward T. Kipreos

发布: 2017年08月05日第7卷第15期 DOI: 10.21769/BioProtoc.2424 浏览次数: 9429

评审: Neelanjan BoseAnonymous reviewer(s)

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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) search Primary culture (原代培养) search Germ stem cells (生殖干细胞) search Tissue culture (组织培养) search

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

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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

干细胞 > 生殖细胞 > 体外培养

细胞生物学 > 细胞分离和培养 > 细胞分离

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