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Dictyostelium Cultivation, Transfection, Microscopy and Fractionation
网炳菌属的培养、转染、显微镜检查和分级分离   

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Abstract

The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. In addition the fractionation of cells and isolation of organelles or known compartments can often verify any subcellular localisation and the use of tagged proteins as bait for the immunoprecipitation of material from cell fractions can identify specific binding partners and multiprotein complexes thereby helping assign a function to the tagged protein. We have successfully applied these techniques to the Dictyostelium discoideum protein TSPOON that is part of an ancient heterohexamer membrane trafficking complex (Hirst et al., 2013). TSPOON is the product of the tstD gene in Dictyostelium and is not required for growth or the developmental cycle in this organism. Dictyostelium amoebae will exist in a vegetative phase where growth is sustained by the phagocytosis of bacteria. When this food source is spent they enter a developmental phase where the amoebae aggregate, via chemotaxis to extracellular waves of cAMP, into multicellular structures that subsequently form a fruiting body containing viable spores (Muller-Taubenberger et al., 2013). In the laboratory this cycle takes less than 24 h to complete and as a further aid to manipulation the requirement for a bacterial food source has been circumvented by the derivatisation of the wild type and isolation of axenic strains that can also grow in a nutrient rich broth. Axenic strains like Ax2 are the mainstay of laboratory research using Dictyostelium (Muller-Taubenberger et al., 2013). A description of Dictyostelium cell cultivation, the generation of cell lines that overexpress TSPOON-GFP and TSPOON null cells, and subsequent analysis (Muller-Taubenberger and Ishikawa-Ankerhold, 2013) is detailed below.

Keywords: Dictyostelium(粘液菌), Cultivation(栽培), Transfection(转染), Microscopy(显微镜), Immunoprecipitation(免疫共沉淀)

Materials and Reagents

  1. Dictyostelium Ax2 cells can be obtained from the Dictyostelium stock centre (http://dictybase.org/StockCenter/StockCenter.html)
    Note: Ax2 strain DBS0235521 is recommended due to the relatively few duplications within its genome (Bloomfield et al., 2008).
  2. HL5 with glucose (Formediun, catalog number: HLG0102 )
  3. LOFLO (Formediun, catalog number: LF1001 )
  4. Ultra-pure dH2O (e.g. Milli-Q system, EMD Millipore)
  5. KH2PO4 (Thermo Fisher Scientific, catalog number: P/4800/53 )
  6. K2HPO4 (anhydrous) (Sigma-Aldrich, catalog number: 60353 )
  7. MgSO4.7H2O (VWR International, catalog number: 25165.260 )
  8. CaCl2.2H2O (VWR International, catalog number: 22317.260 )
  9. HEPES (Sigma-Aldrich, catalog number: H4034 )
  10. KCl (Thermo Fisher Scientific, catalog number: P/4280/53 )
  11. NaCl (VWR International, catalog number: 27810.295 )
  12. NaHCO3 (VWR International, catalog number: 27778.236 )
  13. NaH2PO4.2dH2O (Thermo Fisher Scientific, catalog number: 10723621 )
  14. Na2HPO4 (anhydrous) (Thermo Fisher Scientific, catalog number: S4520/53 )
  15. KOH (VWR International, catalog number: 26668.263 )
  16. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  17. EDTA disodium salt dihydrate (VWR International, catalog number: 20302.260 )
  18. HCl (Thermo Fisher Scientific, catalog number: H/1200/PB08 )
  19. Klebsiella aerogenes (available from http://dictybase.org/StockCenter/StockCenter.html)
  20. 50 mM L-ascorbic acid (Sigma-Aldrich, catalog number: A5960 )
  21. Ethanol (Sigma-Aldrich, catalog number: 3112 )
  22. Chloroform (VWR International, catalog number: 22711.290 )
  23. Plasmids pDT51, pDT61, pDT70 and pJH101 together with gene knockout vectors for iplA, mscS, mclN, phdA and dagA can be obtained from David Traynor
    Note: Plasmid pLPBLP can be ordered through dictyBase (http://www.dictybase.org/db/cgi-bin/dictyBase/SC/plasmid_details.pl?id=9).
  24. HiSpeed® Plasmid Midi Kit (QIAGEN, catalog number: 12643 )
  25. Hygromycin 100 mg/ml sterile solution (Invivogen HygroGold, catalog number: ant-hg-5 )
  26. Blasticidin S 100 mg/ml sterile solution (Invivogen, catalog number: ant-bl-1 )
  27. G418 (20 mg/ml solution) (Life Technologies Geneticin®, catalog number: 11811-031 )
  28. Quick-gDNA MiniPrep kit (Zymo Reasearch, catalog number: D3025 )
  29. Expand 20 kbPLUS PCR system (dNTPack) (F. Hoffmann-La Roche, catalog number: 04743814001 )
  30. Oligonucleotide primers (custom synthesised by Sigma-Aldrich)
  31. Freezing medium [sterilised horse serum containing 7.5% (v/v) DMSO]
  32. Horse serum (Sigma-Aldrich, catalog number: H1270 )
  33. DMSO (Sigma-Aldrich, catalog number: D2650 )
  34. Master stock of 200 mM cAMP
  35. 4x NuPAGE® LDS sample buffer (Life Technologies, Novex, catalog number: NP0008 )
  36. Protein A (PA) sepharose CL-4B beads (GE Healthcare, catalog number: 17-0780-01 )
  37. Acetone (Thermo Fisher Scientific, catalog number: A/0560/17 )
  38. Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 11873580001 )
  39. Precast NuPAGE 4-12% Bis-Tris gels (Life Technologies, Novex)
  40. Coomassie G-250 SimplyBlue safe stain (Life Technologies, InvitrogenTM, catalog number: LC6060 )
  41. PierceTM BCA protein assay (Thermo Fisher Scientific, catalog number: 23225 )
  42. Phenol solution (Equilibrated with 10 mM Tris HCl, pH 8.0, 1 mM EDTA) (Sigma-Aldrich, catalog number: P4557 )
  43. Adenosine 3′, 5′-cyclic monophosphate (cAMP) (free acid) (Sigma-Aldrich, catalog number: A9501 )
  44. TritonTM X-100 solution (~10% in H2O) (Sigma-Aldrich, catalog number: 93443 )
  45. Sodium dodecyl sulphate (SDS) solution (10% in H2O SDS) (Sigma-Aldrich, catalog number: 71736 )
  46. SM agar (Formediun, catalog number: SMA0120 )
  47. SM broth (Formediun, catalog number: SMB0102 )
  48. Trichloroacetic acid (VWR International, catalog number: 102863H )
  49. Q5 (NEB, catalog number: M0491 )
  50. Pwo (Roche Diagnostics, catalog number: 11644947001 )
  51. KOD polymerases (Merk Millipore, catalog number: 71086 )
  52. Antifade mounting medium Fluoromount-G® (Cambridge Bioscience, catalog number: 0100-01 ) 71086 or Prolong® (Life Technologies, catalog number: P10144 )
  53. Axenic medium (see Recipes)
  54. LOFLO medium (see Recipes)
  55. SM agar (see Recipes)
  56. SM broth (see Recipes)
  57. Trichloroacetic acid (see Recipes)
  58. KK2 buffer (see Recipes)
  59. KK2C buffer (see Recipes)
  60. 1 M MgSO4 (see Recipes)
  61. 1 M CaCl2 (see Recipes)
  62. Electroporation buffer E50 (see Recipes)
  63. Dense suspension of Klebsiella aerogenes (see Recipes)
  64. Tris-HCl buffer (see Recipes)
  65. TE buffer (see Recipes)
  66. 20 mg/ml G418 (see Recipes)
  67. Freezing medium (see Recipes)
  68. 50 mM L-Ascorbic (see Recipes)
  69. Master stock of cAMP (see Recipes)
  70. Phosphate buffered saline (PBS) (see Recipes)
  71. PBS-T (see Recipes)
  72. Elution buffer (see Recipes)

Equipment

  1. Sterile tissue culture dishes [(100 x 20 mm style, Corning, catalog number: 353003 or Nunc, catalog number: 172958 ) (35 x 10 mm style, Corning, catalog number: 353001 )]
  2. Sterile 96 flat bottomed well tissue culture dishes (Corning Costar, catalog number: 3799 )
  3. Sterile 50 ml plastic tubes (SARSTEDT AG, catalog number: 62.547.004 or Corning, catalog number: 352070 )
  4. 1.8 ml CryoTube vials (Thermo Fisher Scientific, catalog number: 377267 )
  5. Pipetting reservoir (120 ml capacity) (Thermo Fisher Scientific, catalog number: 10717964 )
  6. Steribag pouches (140 mm x 50 mm x 330 mm) (Thermo Fisher Scientific, catalog number: 12728995 )
  7. Microfuge tubes (Sarstedt Micro tube 1.5 ml) (SARSTEDT AG, catalog number: 72.690 )
  8. Lab-Tek chambered coverglass with cover (Thermo Fisher Scientific, catalog number: 155411 and 155380 )
  9. Glass bottom microwell 35 mm petri dishes with 20 mm microwell (MatTek, catalog number: P35G-1.5-20-C )
  10. Disposable cell spreaders (Biologix, catalog number: 65-100 )
  11. Cryo preservation module (Agilent StratCooler Lite Preservation module, catalog number: 400006 )
  12. 21G (0.8 mm x 40 mm) needles (Becton Dickinson, catalog number: 304432 )
  13. Spatula (150 x 4 mm) (Thermo Fisher Scientific, catalog number: 11503482 )
  14. Platinum wire inoculating loop and insulated holder (Thermo Fisher Scientific, catalog number: 10669652 and 12882775 )
  15. Nunc sterile plastic needles and loops (Thermo Fisher Scientific, catalog number: 254399 and 254410 )
  16. Syringe filters (0.22 µm) for sterilisation of small volumes (Elkay, catalog number: E25-PS22-50S )
  17. Sterile disposable syringes for sterilisation of small volumes (Becton Dickinson, Plastipak 50 ml, catalog number: 30086 ; 20 ml, catalog number: 300613 ; 10 ml, catalog number: 302188 )
  18. Swin-LokTM plastic filter holder 25 mm (GE Healthcare, catalog number: 420200 )
  19. Prefilters. 25 mm cellulose absorbent pads (Merck Millipore, catalog number: AP1002500 )
  20. Nucleopore Track-Etch polycarbonate membrane 3.0 µm (GE Healthcare, catalog number: 110612 )
  21. Micro bio-spin chromatography column (Bio-Rad Laboratories, catalog number: 732-6204 )
  22. Duran bottles (or equivalent autoclavable media bottles) 1,000 ml, 500 ml and 150 ml (Thermo Fisher Scientific, catalog numbers: FB-800-1000 , FB-800-500 and 11699888 )
  23. Foam stoppers (50 mm x 50 mm and 50 mm x 38 mm) (Thermo Fisher Scientific, catalog number: 11522563 and 11512563 )
  24. Aluminium foil (300 mm x 75 mm x 0.02 mm) (Prowrap, catalog number: JR307518U )
  25. Disposable plastic filter bottle units (150 ml) (Millipore Stericup, SCGPU01RE ) or 500 ml (Millipore Stericup, catalog number: SCGPU05RE )
  26. Steritop bottle top filter unit (1,000 ml) (EMD Millipore, SCGPT10RE )
  27. Corning Filter System (1,000 ml) (Corning, catalog number: 430186 )
  28. Universal bottle (Thermo Fisher Scientific, catalog number: BTS-402-050M )
  29. Temperature controlled shaking incubator (e.g. Infors Multitron standard or Innova 4330)
  30. Temperature controlled incubator (e.g. Panasonic, model: MIR-254 PE or MIR-154 PE )
  31. Tissue culture hood (e.g. Scanlaf Mars Safety 2)
  32. Ultrapure water supply (e.g. Milli-Q® Advantage A10 Ultrapure Water Purification System)
  33. Autoclave
  34. Water bath (e.g. Grant SUB Aqua 12 Plus)\
  35. pH meter and calibration buffers (e.g. Hanna Instruments Edge Hybrid pH, catalog number: HI-2020-02 )
  36. Cell counter (e.g. Beckman Coulter Z1) or haemocytometer (Weber Scientific)
  37. Bench top centrifuge (e.g. Thermo Fisher Scientific, model: IEC CL30 or Eppendorf, model: 5702R )
  38. Bench top microfuge (e.g. Eppendorf, model: 5424 or 5424R )
  39. Thermocycler (96 x 0.2 ml tube block) (e.g. Biometra TGradeint or Applied Biosystems Veriti)
  40. Programmable peristaltic pump (e.g. Watson Marlow, model: 505Di )
  41. 8 channel electronic pipette (e.g. eLine 50-1,200 µl) (Sartorius, catalog number: 730391 ) and 50-1,200 µl tips (SafetySpaceTM filter tips) (Sartorius, catalog number: 79121F )
  42. Confocal microscope (e.g. ZEISS, model: LSM710 or LSM780 )
  43. TIRF microscope (e.g. Nikon Corporation)
    Note: Both the Zeiss confocal and Nikon TIRF microscopes are supplied with the manufacturers image analysis software (Zen and NIS-Elements respectively) but due to the limited time users have on this equipment image analysis is usually performed on a dedicated computer work station running these software packages that are an additional purchase from the manufacturers. Free to use image analysis software (open source) packages are also routinely used. ImageJ and Fiji can be used for the confocal and TIRF images. ImageJ can be downloaded from http://imagej.nih.gov/ij and Fiji from http://fiji.sc/Fiji.
  44. Electroporator (e.g. BIO-RAD Xcell) and 1 mm gap width cuvettes (Cell Projects, catalog number: EP-201 )
  45. Potter-Elvehjem homogensier (Wheaton, catalog number: 358049 )
  46. Ultracentrifuge (Beckman Coulter, model: Optima MAX-XP )
  47. Orbitrap Mass Spectrometer (Thermo Fisher Scientific)
  48. 0.2 ml PCR strip tube with 12 wells and separate cap (VWR International, catalog number: 53509-306 )
  49. TLA-110 rotor (Beckman Coulter)
  50. XCell SureLock® Mini-Cell and XCell II™ blot module (Life Technologies, Novex, catalog number: EI0002 ) and power supply (Bio-Rad Laboratories, PowerPacTMHC High-Current Power Supply, catalog number: 164-5052 )

Procedure

  1. Growing Dictyostelium cells
    1. If possible all work with live Dictyostelium discoideum cells should be done in a 22 °C temperature controlled room. The initiation and passaging of cultures either in shaken suspension or in dishes should be done under sterile conditions in a tissue culture hood. It is very important to renew stocks every month from spores or frozen stocks stored at -80 °C or in liquid nitrogen.
    2. Ax2 or the TSPOON null cells (HM1725 and HM1727) cells are grown in shaken suspension (22 °C, 180 rpm) in axenic medium in 250 ml or 500 ml conical flasks (seed at 0.5-2 x 105 cells/ml) and harvested in mid log phase (2-5 x 106 cells/ml). Doubling time in these conditions is normally 8-11 h.
    3.  Alternatively, cells can be grown on tissue culture plates and harvested when confluent (each plate should yield 1-2 x 107 cells) (Figure 1). This is useful when relatively few are required for confocal or TIRF microscopy.


      Figure 1. A phase contrast light photomicrograph of a near confluent lawn of Dictyostelium amoebae. The cells were grown and photographed in a 10 cm tissue culture dish containing 12 ml of axenic medium. Note that there are few gaps between individual cells. The scale is shown in the bottom right.

  2. Electroporation of Dictyostelium cells (based on Pang et al., 1999)
    1. Harvest mid log phase cells into 50 ml sterile plastic tubes and centrifuge at 300 x g for 2-3 min to pellet the cells. Aspirate supernatant and add 50 ml of ice cold E50 buffer and resuspend the cell pellet by gently tapping and shaking the tube.
    2. Determine the number of cells/ml using an automated cell counter or a haemocytometer.
    3. Centrifuge at 300 x g for 2-3 min to pellet the cells and then resuspend at 4 x 107/ml in ice cold E50 buffer. Place the cell suspension on ice and incubate for 5 min (if the cells start to settle then gently mix by agitation).
      1. For overexpression plasmids such as pJH101 (TSPOON-GFP fusion driven by the constitutively active actin15 promoter) or pDT58 and pDT61 (both contain the TSPOON-GFP fusion driven by the TSPOON promoter), transfer 0.1 ml of the cell suspension to a pre-chilled sterile electroporation cuvette (1 mm gap width) and add 10-30 µg of supercoiled plasmid DNA (preferably in ≤15 µl of Tris-HCl or TE buffer and prepared according to the manufacturer’s instructions using the HiSpeed® Plasmid Midi Kit) then mix by gently pipetting up and down avoiding introducing air bubbles to the cell suspension. Return the cuvette to ice (Figure 2).
      2. The most common and reliable gene disruption in Dictyostelium is achieved with blasticidin S as the selective agent using a plasmid containing the resistance cassette (such as pLPBLP) flanked on either side with DNA homologous to the targeted gene (Faix et al., 2004; Faix and Kimmel, 2006). This targeting/disruption cassette should be freed from the remainder of the construct by complete digestion with restriction enzymes. To knockout TSPOON, plasmid pDT70 (based on pLPBLP) was cut with ApaI and SacII (these enzymes generate 3’ overhangs in the cut DNA but enzymes that leave 5’ overahangs e.g. BamHI or blunt cutters like PvuII can be used in any combination to liberate the disruption cassette). The enzymes where removed by a single phenol extraction followed by three extractions with chloroform and the cut DNA precipitated with ethanol (Sambrook and Russel, 2001). Resuspend the cut DNA at 2-3 mg/ml in Tris-HCl or TE buffer. Add 15-20 µg of the disruption cassette DNA to each 0.1 ml of cell suspension, again in a chilled sterile electroporation cuvette (1 mm gap width) and mix as in step B4a.


        Figure 2. A photograph of the electroporation apparatus described in this protocol. In the background the BioRad Xcell is shown with the pod open and loaded with an electroporation cuvette (1cm gap width). In the ice bucket is two more cuvettes awaiting electroporation. In the foreground a tube (15 ml) containing the axenic medium that will be added (0.5 ml) after each cuvette is pulsed twice to electroporate the cell suspension within. A tissue culture dish with 6 wells each of which contains 3 ml of axenic medium is also shown and after recovery on ice for 5 min, 100 µls of the electroporated cell suspension will be added to each well of this dish.

    4. The cell/DNA suspension should be electroporated quickly to minimise cell sedimentation in the cuvette.
      1. Remove the cuvette from the ice and dry off any excess moisture on the electrodes with a tissue before placing in the electroporation pod.
      2. The parameters should be set at 0.75-0.85 kV, 25 µF capacitance with resistance set at infinity (∞) for the Bio-Rad Xcell. The same parameters are used on the Bio-Rad GenePulser and GenePulserII however they may need to be optimised with electroporation devices from other manufacturers.
      3. Deliver two pulses 5 sec apart to each cuvette and then quickly add 0.5 ml of sterile room temperature axenic medium, mix by gentle pipetting up and down and then return the cuvette to ice for 5 min.
      4. For the selection process and screening of transformants see Faix et al. (2004); Kimmel and Faix (2006); Muller-Taubeberger et al. (2006).

  3. Isolation of overexpression and knockout cell lines
    1. For each cuvette prepare a 6 well tissue culture plate with 3 ml of axenic medium in each well.
    2. Remove a cuvette from ice and resuspend the cells by gentle pipetting before adding 0.1 ml to each well of the tissue culture dish.
    3. Incubate at 22 °C overnight (normally 16-24 h) before adding, in duplicate wells, 10 µg/ml, 20 µg/ml and 40 µg/ml G418 (for plasmids pJH101 and pDT61).
    4. For selection with hygromycin, add 30 µg/ml, 60 µg/ml and 90 µg/ml (for pDT58).
    5. Change the selection medium by aspiration to remove dead cells every 2-3 days and the wells should be confluent after 9-14 days. (Hygromycin appears to be less efficient at killing Dictyostelium amoebae than G418 and it is therefore important to replace the medium every 2-3 days as directed to ensure the removal of dead and dying cells that will become detached from the tissue culture plate).
    6. The wells are screened for optimum expression by dislodging the cells by pipetting up and down and removing 0.3 ml into a single well of a chambered coverslip (8 well).
    7. The cells are allowed to settle and attach for 20-30 min before the medium in each well is aspirated and replaced by 0.3 ml of LOFLO medium or KK2C.
    8. The cells are then examined by confocal or TIRF microscopy for the presence and distribution of the GFP fusion protein (Figures 3 and 4).


      Figure 3. Overexpression of TSPOON-GFP. The top panel are cells transfected with pDT61 (TSPOON-GFP fusion protein driven by the Tspoon promoter) and selected for 14 days in axenic medium containing 10µg/ml G418. Most cells express the fusion protein but at varying levels. The bottom panel is mock transfected cells. The cells in both panels where transferred to LoFlo medium for 30 minutes before being imaged by confocal microscopy using exactly the same image collection parameters. Note the faint autofluorescence in the mock transfected cells visible as puncta. The scale is indicated in the bottom right of the figure.


      Figure 4. A TIRF image of TSPOON-GFP. The amoebae were transfected and selected as in Figure 3. Cells were transferred into KK2C (plus 50 µg/ml L-ascorbic acid) 3 h prior to image collection. The scale is indicated.

    9. To make a permanent stock, the well(s) with the optimum expression are harvested by pipetting the axenic medium up and down to dislodge the cells which are then used to initiate a larger culture (grown with the appropriate levels of G418 or hygromycin) either in shaken suspension or several 10 cm tissue culture dishes each with 12 ml of axenic medium.
    10. A minimum of 8 x 107 cells should be harvested and pelleted by centrifugation (300 x g for 2 min) then resuspended in 1.6 ml of freezing medium and processed as in step C23.
    11. To isolate knockout mutants the cell suspension should be diluted to appropriate densities to ensure clonality over a range of cell survival. This is particularly important when the targeted gene affects growth because wild type non-homologous recombinants are likely then to outgrow such a knockout, making the screen and its isolation more difficult.
    12. To ensure clonality, the cell suspension from a single cuvette is first added to a total of 15 ml of axenic medium in 50 ml plastic tube so that the cells are approximately 2.67 x 105/ml.
    13. This cell suspension is further diluted 10 (2.67 x 104/ml), 50 (5.33 x 103/ml), 100 (2.67 x 103/ml), 200 (1.33 x 103/ml) and 400 fold (0.67 x 103/ml). Each dilution is made into a total of 15 ml of axenic medium and 100 µl/well is dispensed into a separate 96 well tissue culture plate for each dilution.
    14. Normally 4 x 96 well plates are prepared for the 100, 200 and 400 fold dilutions so 50 ml of diluted cells should be prepared for each of these dilutions.
    15. Incubate the plates in a moist atmosphere (such as in a large plastic cake storage box lined with wet tissues) at 22 °C overnight (normally 16-24 h) before adding 100 µl of axenic medium containing 20 µg/ml blasticidin S to each well (10 µg/ml final).
    16. Change the selection medium to remove dead cells every 2-3 days by forcefully ‘throwing’ the medium into a large tissue lined (stops splash back into wells) container. Add 200 µl of fresh axenic medium containing 10 µg/ml of blasticidin S to each well.
      Note: Plating out the cells into the 96 well plates and replenishing the medium is best achieved with a 8 channel electronic pipette fitted with tips each with a capacity of 1,200 µl. The cell suspension or fresh media is dispensed from a sterile plastic reservoir. These reservoirs can be reused after autoclaving in a steribag pouch.
    17. The 96 well plate containing the least diluted cells should have confluent wells after 9 days whereas the plates containing the 100, 200 and 400 fold dilutions may take up to 21 days (times are average based on the use of many different knockout vectors but deletion of a gene that impairs growth in axenic medium is likely to increase these times). Dilutions are usually screened from plates where <30 wells/plate are occupied, as these are likely to be clonal.
    18. The cells from each confluent well are harvested by pipetting up and down, then transferred to 1.5 ml microcentrifuge tube containing 0.75 ml of sterile KK2 (this is to dilute residual axenic medium after the cells are pelleted).
    19. Refill the well with 200 µl of fresh axenic medium containing 10 µg/ml of blasticidin S. The cells are pelleted in a benchtop microfuge at full speed for 20 sec and the supernatant removed by aspiration.
    20. Genomic DNA from the cell pellet can then be isolated using a variety of commercial kits but we prefer the Quick-gDNA MiniPrep kit from Zymo Reasearch as it is quick, has few steps and reproducibly yields good quality genomic DNA for screening by PCR using a variety of polymerases [Expand 20 kb plus polymerase (Roche) was found to be optimal for the TSPOON screen but we have also used Q5, Pwo and KOD polymerases].
    21. In the case of the TSPOON knockout, isolation and screening took 17 days from the start of selection until confluent wells where observed in the plates containing the 200 and 400 fold dilutions.
    22. The PCR screen used TSPOON specific primer (TCP) oligonucleotides TCP15: (5’-GATGAAATTTATCAGATATTGATTTCATGAATGTTTCACC-3’) and TCP16 (5’-CTGCTGATGTTGGTTTATAGGTGGCAAACCACCATC-3’) both of which bind outside the disruption cassette to minimise false positives generated by single crossover recombination events. The gene encoding TSPOON (tstD, DDB_G0350235) is located on chromosome 1 and is flanked by genes DDB_G0271002 and DDB_G026990. The exact genomic location of TCP15 is 1:3986383..3986422 and TCP16 is 1:3982321..3982356. All the oligonucleotide primers used in this protocol are located within these coordinates.
      1. The PCR reactions (in 0.2 ml tubes) consisted of a total of 25 µl containing 1x Expand 20kbPLUS buffer, 0.4 nM of each primer, 5 µl of the Quick-gDNA miniprep, 500 µM dNTPs and 0.25 µl (5 units) of Expand 20kbPLUS polymerase.
      2. The cycling parameters were step (1) 92 °C for 1 min 15 sec; (2) 92 °C for 30 sec; (3) 52 °C for 45 sec; (4) 62 °C for 7 min; (5) 62 °C for 5 min. Steps 2 to 4 were repeated a further 36 times before the reactions were held at 4 °C until analysed by gel electrophoresis (Sambrook and Russel, 2001).
      3. The PCR product from wild-type clones is ~4.1 kb and TSPOON knockout clones ~4.7 kb.
      4. The insertion of blasticidin resistance cassette and flanking DNA introduces restriction sites into the knockout PCR product that are absent in the wild type. This is a useful further proof of correct targeting as digestion of these products with a restriction enzyme such as SmaI for TSPOON, leaves the wild type product intact but cuts the knockout product into 3 fragments.
      5. A total of 46 wells where screened by this method, 37 were TSPOON knockouts, 6 were wild type and 3 were not clonal as they contained both PCR products. Therefore, the targeting frequency for this the tstD gene was 80% and though high was in keeping with other genes targeted by this procedure in our laboratory such as 76% for iplA, 67% mscS, 57% mclN, 50% phdA and 12% dagA.
    23. Four TSPOON knockouts, isolated from different 96 well plates, were processed further (2 for study and 2 as backup).
      1. The wells containing these cells were harvested as before, the cells diluted in KK2 to 0.6-1.2 cells/µl (a confluent well contains ~105 cells).
      2. SM agar plates were prepared each with a 0.4 ml drop of a dense suspension of Klebsiella aerogenes bacteria (Figure 5).


        Figure 5. A SM agar plate covered in a lawn of Klebsiella aerogenes with Dictyostelium colonies visible. The translucent periphery of a colony consists of vegetative amoebae while at the centre fruiting bodies and other multicellular structures have formed. Colonies suitable for harvesting and freezing should be ~1 to 1.5 cm in diameter and clearly separated from neighbours. Suitable colonies in this example are indicated by the black arrowheads. Approximately 6 amoebae in 10 µl of KK2 were directly added a 400 µl drop of a dense suspension of Klebsiella aerogenes already on the plate and then evenly spread. Colonies were visible 4 days after plating and this photograph was taken after 6 days. The scale is shown in the bottom right.

      3. Add 10 µl of the diluted cell suspension to this drop and spread evenly over the plate with a sterile plastic spreader.
      4. Discrete colonies appear within 4-5 days and one per knockout was harvested when they had reached ~1.5 cm in diameter.
      5. The flat end of a sterilised small metal spatula was used to transfer the entire colony directly into 1.6 ml of freezing medium within a CryoTube on ice. The CryoTubes were intermittently vortexed to ensure an even suspension and then transferred to cryo preservation module chilled to 4 °C.
      6.  The module is then transferred to a -80 °C freezer overnight and then the tubes can remain at -80 °C (cells remain viable for >2 years) or be transferred to a liquid nitrogen storage tank (indefinite viability).

  4. Microscopy
    1. Fixed and live cells were analysed my microscopy at the vegetative or the aggregation competent stage of development.

      For live vegetative cells
      1. Simply harvest growing cells from axenic culture and transfer an appropriate number of cells so that final density is 0.5-1.5 x 105/cm2 into a glass bottom dish or chambered coverslip containing LOFLO medium (this has been formulated to minimise background and cellular autofluorescence compared to standard axenic medium and it will keep the cells vegetative for a while) or KK2C (they will start to develop in this buffer but autofluorescence and photosensitivity will be minimised).
      2. Incubate at 22 °C for 10 min to allow the cells to attach then aspirate the medium (do not allow the cells to dry) and replace with fresh LOFLO or KK2C. They can then be used for live cell imaging or fixed.

      For developed cells
      1. Transfer 0.5-1.5 x 105/cm2 into KK2C and replace the KK2C once the cells have attached.
      2. Incubate at 22 °C for 8-10 h to allow the cells to become aggregation competent.
      3. Alternatively transfer 1 x 106 cells into a 35 mm tissue culture dish containing 2 ml of KK2C, allow the cells to attach and then replace the KK2C and incubate at 22 °C for 1 h.
      4. Transfer the dish to 15 °C overnight (15-17 h) and then return the dish to 22 °C for 1 h before transfer (at 0.5-1.5 x 105/cm2) to a glass bottom dish (the cells should aggregate within 2-3 h at 22 °C).
      5. If larger quantities of developed cells are needed then resuspend vegetative cells at 2 x 107/ml in KK2C and transfer them into a conical flask with a volume of at least 5 times that of the cell suspension to ensure optimum aeration. The cells are shaken at 180 rpm at 22 °C for 1 h then a 100 µl droplet of KK2C, containing enough cAMP (diluted from a master stock) so that the final concentration of the entire suspension is 100 nM, is dispensed into the suspension every six minutes for 3.5-5.0 h via a programmable peristaltic pump. This procedure mimics the pulsatile waves of cAMP that are emitted through a developing population of amoebae and is useful for mutant strains defective in early development and cAMP signalling.
      6. Harvest the cells and pellet by centrifugation 300 x g for 2 min, aspirate off the supernatant and resuspend the cells at 1 x 107/ml in KK2C and plate as before at 0.5-1.5 x 105/cm2.
      7. There are numerous ways to fix Dictyostelium amoebae for immunocytochemistry (ICC) and they can be found elsewhere (DictyBase1 and 2, Jungbluth et al., 1994; Hagedorn et al., 2006).
    2. Confocal laser scanning microscopy.
      1. Live cell imaging is normally a balance between detecting the tagged fusion protein (signal to noise ratio) and damaging the cells with the laser due to free radicals produced by the illumination interacting with cellular constituents or the fluorophore tag. With a 10x or 20x lens this is never normally a problem but at these magnifications there is little or no spatial resolution within a cell to be gained given that Dictyostelium cells are ~10-15 µm in diameter.
      2. Normally a 63x or 100x lens is needed to resolve the subcellular localisation of a tagged protein within a Dictyostelium amoeba.
      3. Vegetative amoebae are particularly sensitive to damage especially from shorter wavelengths such as the 405 nm laser. An Argon laser (25-35 mW) is fitted to most confocal miscroscopes for imaging YFP, GFP and CFP fusion proteins and excessive power will result in phototoxicity and photobleaching so the laser strength should be kept to a minimum (normally 2-10% for the 488 nm laser line).
      4. Longer wavelength laser lines such as 561 mn used for RFP tagged proteins present less of a problem and can be used at higher power (>15%) signal can be increased by opening the pinhole.
      5. Avoid line averaging, slow scans and real time deconvolution.
      6. Developed cells are usually less sensitive to laser damage but it can be fusion protein dependent. For instance a GFP tagged protein that resides in the plasma membrane may render the cells more sensitive (due to lipid oxidation in the membrane) than a cytoplasmic protein. L-Ascorbic acid (50-100 µm final) to scavenge free radicals can be added to minimise this problem and the vitamin E analogue Trolox C has also been used to this end (add 1 mM to the growth medium and incubate overnight before imaging).
      7. If phototoxicity cannot be overcome then consider switching to a spinning disc confocal microscope (SDCM) where illumination is restricted to thousands of small confocal volumes during image acquisition rather than through the whole sample thus reducing photobleaching and phototoxicity.
      8. To follow the movement of vegetative cells frames should be captured every 2-30 sec and for developed cells every 1-20 sec. The fastest realistic frame rate is 1/sec. It may be necessary to reduce the area scanned from the default 512 pixels x 512 pixels to 512 x 300 as this allows slower scans (better images) and maximises the frame rate (Frigault et al., 2009; Muller-Taubenberger and Ishikawa-Ankerhold, 2011).
      9.  For cells expressing the promoter_TSPOON-GFP construct typical settings used had the 488 nm laser power set at 5% and the pinhole opened up to 2.5 Airy units with a 512 pixels x 300 pixels frame collected every second. For faster temporal resolution switch to a SDCM.
      10.  With fixed cells and ICC photobleaching can be a problem but the use of an antifade mounting medium (typically Fluoromount-G®) can minimise this and allows slower scan speeds and averaging to give better images.
    3. TIRF microscopy.
      1. The problem of phototoxicity is usually more acute with vegetative Dictyostelium cells, in part because although the evanescent field is limited to the initial ~100 nm of the sample from the coverslip, the plasma membrane is strongly illuminated.
      2. Image cells that have been in LOFLO or KK2C for at least one hour. The inclusion of L-Ascorbic acid may be necessary.
      3. Keep the laser power and the illumination time to a minimum.
      4. Since there is no scanning the frame rate is higher. A 100x lens (high NA) should be used with an additional 1.5x zoom lens if present as this allows ameobae to be imaged in great detail.
      5. Filter all buffers (0.22 µm) to remove particulates.
      6. Typical settings for cells expressing promoter_TSPOON-GFP construct had the 488 nm laser power set at 7% with an exposure time of 0.08 sec collecting 512 x 512 frames at up to 12 per sec.

  5. Cell Fractionation
    1. Cells expressing A15_GFP (control) or promoter_TSPOON-GFP were grown until they reached a density of 2-4 x 106/ml in selective media, and by microscopy >50% of cells were expressing GFP.
    2. Starting with a maximum of 8 x 108 cells, the cells were washed in KK2 buffer and then pelleted at 300 x g for 3 min. All subsequent steps were performed at 4 °C.
    3. The cells were resuspended in PBS with a protease inhibitor cocktail, lysed by 8 strokes of a motorized Potter–Elvehjem homogenizer (grinding chamber clearance 0.1-0.15 mm) followed by 5 strokes through a 21-g needle to ensure full lysis.
    4. Alternatively, the cells can be resuspended at 1-5 x 108/ml and placed in a syringe fitted with a 25 mm filter holder containing a prefilter and a Nucleopore filter (3.0 µm). The cell suspension is then passed through this filter assembly twice.
    5. The lysate was then centrifuged at 4,100 x g for 32 min to pellet nuclei and unbroken cells, and the postnuclear supernatant further centrifuged at 50,000 rpm (135,700 x g RCFmax) for 30 min in a TLA-110 rotor to recover the membrane pellet and cytosolic supernatant.
    6. A standard protein assay was used to assess protein recovery in the 2 fractions, and volumes adjusted for equal protein.
    7. Alternatively, to equalise volumes, the cytosolic supernatant was concentrated by precipitation with 10% tricholoroacetic acid at 4 °C for 30 min and recovered by centrifugation 14,000 x g for 10 min. Samples precipitated with trichloroacetic acid were washed with ice cold acetone (-20 °C), air dried for 2 min and then resuspended in the same volume as the pellet fractions.

  6. Protein pulldowns
    Immunoprecipitations were performed using amoebae stably expressing TSPOON-GFP under a constitutive (A15_ TSPOON-GFP) and its own promoter (prom_TSPOON-GFP), and non-transformed cells were used as a control. The amounts given here are recommended for a large scale immunoprecipitation sufficient for proteomic identification of interacting proteins under native conditions. The size of the starting culture, and all subsequent volumes can be reduced accordingly for smaller scale immunoprecipitations, for instance where the identification of proteins are made by Western blotting. The protocol can also be amended for denatured (by heat and/or the presence of SDS but are not detailed here) immunoprecipitations.
    1. Cells were grown until they reached a density of 2-4 x 106/ml in selective media, and by microscopy >50% of cells were expressing GFP.
    2. Up to 8 x 108 cells were pelleted by centrifugation at 300 x g for 2 min, washed twice in 50 ml of KK2 buffer before being resuspended at 2 x 107 cells/ml in KK2 buffer and starved for 4-6 h at 22 °C whilst shaking at 180 rpm.
    3. The cells were then pelleted at 300 x g for 3 min, lysed in 4 ml PBS-T plus protease inhibitor cocktail tablet, extracted for 20 min with rotation at 8 rpm in a 15 ml tube at 4 °C, and then spun 20,000 x g for 15 min to remove debris and insoluble material.
    4. By BCA protein assay the resulting lysate contained 10-15 mg total protein.
    5. The lysates were precleared by adding 100 µl of PA sepharose bead slurry (50% v/v in PBS) and incubated for 30 min with rotation at 8 rpm, followed by centrifugation at 2,200 x g for 3 min to pellet beads.
    6. The supernatant was transferred (~ 5 ml) to a fresh tube, and this is the starting material for the immunoprecipitation (protein concentration should be 2-4 mg/ml).
    7. For maximum recovery the lysates were immunoprecipitated using an in-house antibody against GFP overnight with rotation at 4 °C, though it may be sufficient to incubate for as little as 90 min.
    8. The appropriate antibody concentration requires individual optimization, though the recommended starting point is generally 2 to 5-fold higher than used for Western blotting.
    9.  Following incubation with anti-GFP, 50 µl PA sepharose bead slurry (50% v/v in PBS) was added for 90 min at 4 °C with rotation at 8 rpm.
    10. An alternative would be to use a commercial source of polyclonal anti-GFP, or an anti-GFP that is already coupled to sepharose, for example GFP-TRAP that has the benefit of the antibody remaining coupled to the PA sepharose when immunoprecipitated proteins are eluted from the beads. In this case the addition of PA sepharose at this step is omitted.
    11. The antibody complexes recovered by centrifugation 2,200 x g for 3 min, and then washed twice with 10 ml PBS-T, resuspended in 1 ml of PBS, and transferred into a 1.5 ml microfuge tube.
    12.  The antibody complexes were washed a further two times with PBS, pelleting the beads at 8,000 x g for 20 sec, and then eluted from the beads with 200 µl elution buffer warmed to 60 °C for 10 min.
    13. The beads are then pelleted at 8,000 x g for 20 sec, and the supernatant carefully removed; this can be facilitated by using a fine gel loading tip to remove the last 50 µl as the beads do not enter the tip, or by the use a micro bio-spin chromatography column.
    14. Eluted proteins were then precipitated with 1.2 ml acetone overnight (3-20 h) at -20 °C and centrifuged at 10,000 x g for 5 min, 4 °C. An overnight precipitation increases the yield particularly for low protein concentrations but extending it beyond this results in a lower recovery.
    15. The supernatant is removed and the pellet air dried for 2 min and resuspended in a buffer of choice (e.g. 1x LDS sample buffer for SDS-PAGE).
    16. For Western blotting of samples SDS-PAGE gels were run and transferred according to a standard protocol (Sambrook and Russel, 2001).
    17. For proteomics, the samples were run on pre-cast NuPAGE 4-12% Bis-Tris gels, stained with Coomassie G-250 SimplyBlue SafeStain and then cut into 8 gel slices. Each gel slice was processed by filter-aided sample preparation solution digest, and the sample was analyzed by liquid chromatography–tandem mass spectrometry in an Orbitrap mass spectrometer.
    18. Proteins that came down in the non-transformed control were eliminated, as were any proteins with less than 5 identified peptides, proteins that did not consistently coimmunoprecipitate in three independent experiments, or proteins of very low abundance compared with the bait (i.e., molar ratios of <0.002). The remaining proteins were considered to be specifically immunoprecipitated.

Recipes

  1. Axenic medium
    35.5 g HL5 with glucose
    200 mg dihydrostreptomycin
    Note: Do not use streptomycin as it is inactivated during autoclaving.
    Add ultrapure dH2O to 1,000 ml
    Dispense into conical flasks
    75 ml/250 ml flask stoppered with 50 mm x 38 mm foam bung
    150 ml/500 ml flask stoppered with 50 mm x 38 mm foam bung
    750 ml/ 2,000 ml flask stoppered with 50 mm x 50 mm foam bung
    The top of each flask should be covered over with a small piece (12 cm x 12 cm) of aluminium foil to prevent the top of the stopper being contaminated by dust during storage
    Autoclave to sterilise
    Note: 121 °C for no longer than 15 min and remove from the autoclave as soon as possible to avoid caramelisation i.e. the media turning dark brown in colour. With larger more sophisticated autoclaves with extra safety features that extend the cycle time then this may have to be reduced to as low as 7 min.
    Alternatively filter sterilise (Steritop 1,000 ml unit, 0.22 µm) into sterile 1,000 ml Duran bottles or use a 1,000 ml Corning filter unit (0.22 µm)
    Stored in the dark at 22 °C
  2. LOFLO medium
    8.4 g LOFLO medium
    100 mg dihydrostreptomycin
    Add ultrapure dH2O to 500 ml
    Filter sterilise (0.22 µm) using a 500 ml Stericup filter unit
    Store in the dark at 22 °C (short term) or 4 °C (long term)
  3. SM agar
    41.7 g SM agar
    Add ultrapure dH2O to 1,000 ml
    Autoclave to sterilise
    Note: 121 °C for no longer than 15 min and remove from the autoclave as soon as possible to avoid caramelisation. With larger more sophisticated autoclaves with extra safety features that extend the cycle time then this may have to be reduced to as low as 7 minutes.
    Cool to 60 °C in a heated water bath prior to pouring 25-30 ml per 10 cm diameter plastic petridish
    Once the agar has set and cooled place the dishes in an airtight box or seal up in batches (15-20 plates) in plastic bags (use the bags the dishes were packed in) to minimise desiccation
    Stored in the dark at 22 °C (short term <1 week) or 4 °C (long term)
  4. SM broth
    24.7 g SM broth
    Add ultrapure dH2O to 1,000 ml
    Dispense 10 ml into each 20 ml Universal bottle
    Autoclave to sterilise
    Note: 121 °C for no longer than 15 min and remove from the autoclave as soon as possible to avoid caramelisation. With larger more sophisticated autoclaves with extra safety features that extend the cycle time then this may have to be reduced to as low as 7 min.
    Stored in the dark at 22 °C
  5. KK2 buffer
    2.24 g KH2PO4
    0.52 g K2HPO4, anhydrous
    Add ultrapure dH2O to 1,000 ml
    Autoclave to sterilise in 1,000 ml Duran bottles (121 °C for 15 min)
    pH after autoclaving should be ~6.1
    Add 2 ml of sterile 1 M MgSO4
    Stored at 22 °C
  6. KK2C buffer
    1,000 ml of sterile KK2
    0.1 ml of sterile 1 M CaCl2
    Stored at 22 °C
  7. 1 M MgSO4
    246.48 g MgSO4
    Add ultrapure dH2O to 1,000 ml
    Autoclave to sterilise in 150 ml Duran bottles (121 °C for 15 min)
    Stored at 22 °C
  8. 1 M CaCl2
    147.02 g CaCl2
    Add ultrapure dH2O to 1,000 ml
    Autoclave to sterilise in 150 ml Duran bottles (121 °C for 15 min)
    Stored at 22 °C
  9. Dense suspension of Klebsiella aerogenes
    Prepare a SM agar plate with Klebsiella streaked out on it so that there are individual colonies (stored at 8 °C and renew every 2 months from frozen stocks)
    Pick a colony with a flame sterilised platinum loop (or a disposable sterile plastic needle or plastic loop) and use this to seed a universal bottle containing SM broth
    Incubate the bottle for 48 h at 22 °C and vortex before using
    Stored in the dark at 8 °C
    Renew every month
  10. Electroporation buffer E50
    2.38 g HEPES
    1.86 g KCl
    0.29 g NaCl
    0.5 ml of 1 M MgSO4
    0.21 g NaHCO3
    0.08 g NaH2PO4.2dH2O
    Add ultrapure dH2O to 500 ml
    Adjust to pH 7.0 with KOH
    Filter sterilise (0.22 µm) using a 500 ml Stericup filter unit and stored at 4 °C
  11. Tris-HCl buffer
    0.12 g Trizma® base
    Add ultrapure dH2O to 100 ml
    Adjust to pH 8.0 with HCl
    Autoclave to sterilise in a 150 ml Duran bottle
    Stored at 22 °C
  12. TE buffer
    0.12 g Trizma® base
    0.04 g EDTA
    Add ultrapure dH2O to 100 ml
    Adjust to pH 8.0 with HCl
    Autoclave to sterilise in a 150 ml Duran bottle
    Stored at 22 °C
  13. 20 mg/ml G418
    552 mg Geneticin® G418 sulphate adjusted for a potency of 724 µg/mg
    Note: Potency varies from batch to batch.
    Add ultrapure dH2O to 20 ml
    Filter sterilise with 20 ml syringe fitted with a 0.22 µm syringe filter
    Stored at -20 °C in 1 ml aliquots in sterile 1.5 ml microfuge tubes the G418 solution retains its potency for >2 years. Working stocks should be stored at 4oC and used within 6 months
  14. Freezing medium
    150 ml horse serum
    11.25 ml [7.5% (v/v)] DMSO
    Filter sterilise (0.22 µm) using a 150 ml Stericup filter unit
    Aliquot into sterile 50 ml tubes and stored at -20 °C and should be used within 1 year
  15. 50 mM L-Ascorbic
    0.22 g L-Ascorbic
    Add ultrapure dH2O to 25 ml
    Filter with 50 ml syringe fitted with a 0.22 µm syringe filter to remove particulates
    Stored in 0.5 ml aliquots at -20 °C
  16. Master stock of cAMP
    6.6 g cAMP
    Add ultrapure dH2O to 95 ml
    Adjust to pH 7.0 very carefully with KOH (Note: All the cAMP will not dissolve until the pH <5)
    Add ultrapure dH2O to final volume of 100 ml
    Filter sterilise (0.22 µm) using a 150 ml Stericup filter unit and stored in 20 ml aliquots at -20 °C
  17. PBS
    8.01 g NaCl
    0.20 g KCl
    1.42 g Na2HPO4, anhydrous
    0.24 g KH2PO4
    Add ultrapure dH2O to 1,000 ml
    Autoclave in 500 ml Duran bottles to sterilise (121 °C for 15 min)
    Stored at 22 °C
  18. PBS-T
    PBS adjusted to 1% Triton X-100, using a 10% Triton stock solution
    Filter sterilise for long term storage (0.22 µm)
    Stored at 22 °C or 4 °C (long term storage)
  19. Trichloroacetic acid
    100 g trichloroacetic acid
    Dissolve in 35 ml ultrapure dH2O
    Then make up to 100ml with ultrapure dH2O
    Stored at 22 °C
  20. Elution buffer
    1.21 g Trizma® base
    Add ultrapure dH2O to 75 ml
    20 ml SDS 2% (w/v) from 10% solution
    Adjust to pH 8.0 with HCl
    Add ultrapure dH2O to a final volume of 100 ml
    Filter sterilise (0.22 µm) using a 150 ml Stericup filter unit and stored in 1 ml aliquots at
    -20 °C

Acknowledgments

This study was supported by the Medical Research Council [(MC_U105115237), DT and RRK] and the The Wellcome Trust (JH) .The electroporation of Dictyostelium amoebae is based on the method of Pang, Lynes and Knecht (Pang et al., 1999).

References

  1. Bloomfield, G., Tanaka, Y., Skelton, J., Ivens, A. and Kay, R. R. (2008). Widespread duplications in the genomes of laboratory stocks of Dictyostelium discoideum. Genome Biol 9(4): R75.
  2. DictyBase 1 http://www.dictybase.org/techniques/geneex/indirect_immunofluo.html.
  3. DictyBase 2 http://www.dictybase.org/techniques/microscopy/fixation_rgomer.html.
  4. Faix, J., Kreppel, L., Shaulsky, G., Schleicher, M. and Kimmel, A. R. (2004). A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system. Nucleic Acids Res 32(19): e143.
  5. Hagedorn, M., Neuhaus, E. M. and Soldati, T. (2006). Optimized fixation and immunofluorescence staining methods for Dictyostelium cells. Methods Mol Biol 346: 327-338.
  6. Hirst, J., Schlacht, A., Norcott, J. P., Traynor, D., Bloomfield, G., Antrobus, R., Kay, R. R., Dacks, J. B. and Robinson, M. S. (2014). Characterization of TSET, an ancient and widespread membrane trafficking complex. Elife 3: e02866.
  7. Jungbluth, A., von Arnim, V., Biegelmann, E., Humbel, B., Schweiger, A. and Gerisch, G. (1994). Strong increase in the tyrosine phosphorylation of actin upon inhibition of oxidative phosphorylation: correlation with reversible rearrangements in the actin skeleton of Dictyostelium cells. J Cell Sci 107 ( Pt 1): 117-125.
  8. Kimmel, A. R. and Faix, J. (2006). Generation of multiple knockout mutants using the Cre-loxP system. Methods Mol Biol 346: 187-199.
  9. Muller-Taubenberger, A. (2006). Application of fluorescent protein tags as reporters in live-cell imaging studies. Methods Mol Biol 346: 229-246.
  10. Muller-Taubenberger, A. and Ishikawa-Ankerhold, H. C. (2013). Fluorescent reporters and methods to analyze fluorescent signals. Methods Mol Biol 983: 93-112.
  11. Muller-Taubenberger, A., Kortholt, A. and Eichinger, L. (2013). Simple system--substantial share: the use of Dictyostelium in cell biology and molecular medicine. Eur J Cell Biol 92(2): 45-53.
  12. Pang, K. M., Lynes, M. A. and Knecht, D. A. (1999). Variables controlling the expression level of exogenous genes in Dictyostelium. Plasmid 41(3): 187-197.
  13. Sambrook, J. and Russel, D. W. (2001). Molecular cloning: A laboratory manual. Third edition. Cold Spring Harbor.

简介

使用更复杂的显微镜,活细胞中荧光标记的蛋白质的实时可视化大大增加了我们对基本生理过程如细胞运动,趋化性,细胞分裂和膜运输过程中关键蛋白质动力学的了解。此外,细胞的分级和分离细胞器或已知的隔室通常可以验证任何亚细胞定位,并且使用标记的蛋白质作为诱饵用于来自细胞部分的物质的免疫沉淀可以鉴定特异性结合配偶体和多蛋白复合物,从而有助于赋予功能标记蛋白。我们已经成功地将这些技术应用于作为古代异构六聚体膜转运复合物的一部分的盘基网柄菌discoideum蛋白TSPOON(Hirst等,2013)。 TSPOON是盘根杆菌中tstD基因的产物,不需要生物体或发育周期。盘根盘菌属将存在于通过细菌吞噬而持续生长的营养期。当这种食物来源消耗时,它们进入发育阶段,其中的变形虫通过趋化因子到细胞外的cAMP波形聚集成多细胞结构,随后形成含有活孢子的果实体(Muller-Taubenberger等,2013)。在实验室中,这个循环需要不到24小时才能完成,并且作为进一步的辅助操作,对细菌食物来源的要求已经通过野生型的衍生化和也可以在富含营养的肉汤中生长的无性菌株的分离来规避。像Ax2这样的轴突菌株是使用盘基网柄菌的实验室研究的支柱(Muller-Taubenberger等,2013)。盘根杆菌细胞培养的描述,过表达TSPOON-GFP和TSPOON无效细胞的细胞系的产生以及随后的分析(Muller-Taubenberger和Ishikawa-Ankerhold,2013)的描述如下。

关键字:粘液菌, 栽培, 转染, 显微镜, 免疫共沉淀

材料和试剂

  1. Axic细胞可以从盘基网柄菌种中心获得( http://dictybase.org/StockCenter/StockCenter.html
    注意:Ax2菌株DBS0235521是推荐的,因为其基因组中的重复相对较少(Bloomfield等人,2008)。
  2. HL5与葡萄糖(Formediun,目录号:HLG0102)
  3. LOFLO(Formediun,目录号:LF1001)
  4. 超纯的dH 2 O(例如Milli-Q系统,EMD Millipore)

  5. (Thermo Fisher Scientific,目录号:P/4800/53)
  6. (无水)(Sigma-Aldrich,目录号:60353)
  7. MgSO 4·7H 2 O(VWR International,目录号:25165.260)。
  8. (VWR International,目录号:22317.260)。
  9. HEPES(Sigma-Aldrich,目录号:H4034)
  10. KCl(Thermo Fisher Scientific,目录号:P/4280/53)
  11. NaCl(VWR International,目录号:27810.295)
  12. NaHCO 3(VWR International,目录号:27778.236)
  13. (Thermo Fisher Scientific,目录号:10723621)的二硫化物和二硫代氨基甲酸盐的反应产物。
    2
  14. (无水)(Thermo Fisher Scientific,目录号:S4520/53)
  15. KOH(VWR International,目录号:26668.263)
  16. (Sigma-Aldrich,目录号:T1503)
  17. EDTA二钠盐二水合物(VWR International,目录号:20302.260)
  18. HCl(Thermo Fisher Scientific,目录号:H/1200/PB08)
  19. 克雷伯菌(Klebsiella aerogenes)(可从 http://dictybase.org/StockCenter/StockCenter.html
  20. 50mM L-抗坏血酸(Sigma-Aldrich,目录号:A5960)
  21. 乙醇(Sigma-Aldrich,目录号:3112)
  22. 氯仿(VWR International,目录号:22711.290)
  23. 将质粒pDT51,pDT61,pDT70和pJH101与用于iplA ,mscS ,mclN , phdA 的基因敲除载体 dagA 可以从David Traynor获得
    注意:质粒pLPBLP可以通过dictyBase进行排序( http://www.dictybase.org/db/cgi-bin/dictyBase/SC/plasmid_details.pl?id=9 )。
  24. HiSpeed ® Plasmid Midi Kit(QIAGEN,目录号:12643)
  25. 潮霉素100mg/ml无菌溶液(Invivogen HygroGold,目录号:ant-hg-5)
  26. 杀稻瘟素S 100mg/ml无菌溶液(Invivogen,目录号:ant-b1-1)
  27. G418(20mg/ml溶液)(Life Technologies Geneticin ,目录号:11811-031)
  28. Quick-gDNA MiniPrep试剂盒(Zymo Reasearch,目录号:D3025)
  29. 扩增20kb PLUS PCR系统(dNTPack)(F.Hoffmann-La Roche,目录号:04743814001)
  30. 寡核苷酸引物(由Sigma-Aldrich定制合成)
  31. 冷冻培养基[含7.5%(v/v)DMSO的灭菌马血清]
  32. 马血清(Sigma-Aldrich,目录号:H1270)
  33. DMSO(Sigma-Aldrich,目录号:D2650)
  34. 200mM cAMP的母料
  35. 4x NuPAGE LDS样品缓冲液(Life Technologies,Novex,目录号:NP0008)
  36. 蛋白A(PA)琼脂糖CL-4B珠(GE Healthcare,目录号:17-0780-01)
  37. 丙酮(Thermo Fisher Scientific,目录号:A/0560/17)
  38. 蛋白酶抑制剂混合物(F.Hoffmann-La Roche,目录号:11873580001)
  39. Precast NuPAGE 4-12%Bis-Tris凝胶(Life Technologies,Novex)
  40. 考马斯G-250 SimplyBlue安全染色剂(Life Technologies,Invitrogen TM ,目录号:LC6060)
  41. Pierce BCA蛋白测定(Thermo Fisher Scientific,目录号:23225)
  42. 苯酚溶液(用10mM Tris HCl,pH8.0,1mM EDTA平衡)(Sigma-Aldrich,目录号:P4557)
  43. 腺苷3',5'-环一磷酸(cAMP)(游离酸)(Sigma-Aldrich,目录号:A9501)
  44. Triton X-100溶液(〜10%,在H 2 O中)(Sigma-Aldrich,目录号:93443)
  45. 十二烷基硫酸钠(SDS)溶液(10%于H 2 O SDS中)(Sigma-Aldrich,目录号:71736)
  46. SM琼脂(Formediun,目录号:SMA0120)
  47. SM肉汤(Formediun,目录号:SMB0102)
  48. 三氯乙酸(VWR International,目录号:102863H)
  49. Q5(NEB,目录号:M0491)
  50. Pwo(Roche Diagnostics,目录号:11644947001)
  51. KOD聚合酶(Merk Millipore,目录号:71086)
  52. 防褪色封固剂Fluoromount-G(Cambridge Bioscience,目录号:0100-01)71086或Prolong (Life Technologies,目录号:P10144)
  53. 中等(见配方)
  54. LOFLO介质(见配方)
  55. SM琼脂(见配方)
  56. SM肉汤(请参阅食谱)
  57. 三氯乙酸(参见配方)
  58. KK 2 缓冲区(请参阅配方)
  59. KK 2 C缓冲区(参见配方)
  60. 1 M MgSO 4(参见配方)
  61. 1 M CaCl <2> (参见配方)
  62. 电穿孔缓冲液E50(参见配方)
  63. 浓缩克雷伯菌(Klebsiella aerogenes)的悬浮液(见配方)
  64. Tris-HCl缓冲液(见配方)
  65. TE缓冲区(参见配方)
  66. 20mg/ml G418(见Recipes)
  67. 冻结介质(见配方)
  68. 50 mM L-抗坏血酸(见配方)
  69. cAMP的主库存(见配方)
  70. 磷酸盐缓冲盐水(PBS)(见Recipes)
  71. PBS-T(参见配方)
  72. 洗脱缓冲液(参见配方)

设备

  1. 将无菌组织培养皿[(100×20mm型,Corning,目录号:353003或Nunc,目录号:172958)(35×10mm型,Corning,目录号:353001)
  2. 无菌96平底孔组织培养皿(Corning Costar,目录号:3799)
  3. 无菌50ml塑料管(SARSTEDT AG,目录号:62.547.004或Corning,目录号:352070)
  4. 1.8ml CryoTube小瓶(Thermo Fisher Scientific,目录号:377267)
  5. 移液容器(120ml容量)(Thermo Fisher Scientific,目录号:10717964)
  6. Steribag袋(140mm×50mm×330mm)(Thermo Fisher Scientific,目录号:12728995)
  7. 微量离心管(Sarstedt Micro tube 1.5ml)(SARSTEDT AG,目录号:72.690)
  8. 带盖的Lab-Tek带盖玻片(Thermo Fisher Scientific,目录号:155411和155380)
  9. 带有20mm微孔的玻璃底微孔35mm培养皿(MatTek,目录号:P35G-1.5-20-C)
  10. 一次性细胞涂布器(Biologix,目录号:65-100)
  11. 冷冻保存模块(Agilent StratCooler Lite Preservation module,目录号:400006)
  12. 21G(0.8mm×40mm)针(Becton Dickinson,目录号:304432)
  13. 刮刀(150×4mm)(Thermo Fisher Scientific,目录号:11503482)
  14. 铂丝接种环和绝缘支架(Thermo Fisher Scientific,目录号:10669652和12882775)
  15. Nunc无菌塑料针和环(Thermo Fisher Scientific,目录号:254399和254410)
  16. 用于小体积灭菌的注射器过滤器(0.22μm)(Elkay,目录号:E25-PS22-50S)
  17. 用于小体积灭菌的无菌一次性注射器(Becton Dickinson,Plastipak 50ml,目录号:30086; 20ml,目录号:300613; 10ml,目录号:302188)
  18. Swin-Lok TM 塑料过滤器支架25mm(GE Healthcare,目录号:420200)
  19. 预过滤器。 25mm纤维素吸收垫(Merck Millipore,目录号:AP1002500)
  20. Nucleopore Track-Etch聚碳酸酯膜3.0μm(GE Healthcare,目录号:110612)
  21. 微生物自旋色谱柱(Bio-Rad Laboratories,目录号:732-6204)
  22. (Thermo Fisher Scientific,目录号:FB-800-1000,FB-800-500和11699888)的杜兰瓶(或等同的可高压灭菌培养基瓶)
  23. 泡沫塞(50mm×50mm和50mm×38mm)(Thermo Fisher Scientific,目录号:11522563和11512563)
  24. 铝箔(300mm×75mm×0.02mm)(Prowrap,目录号:JR307518U)
  25. 一次性塑料过滤瓶单元(150ml)(Millipore Stericup,SCGPU01RE)或500ml(Millipore Stericup,目录号:SCGPU05RE)
  26. Steritop瓶顶过滤器单元(1000ml)(EMD Millipore,SCGPT10RE)
  27. Corning过滤系统(1,000ml)(Corning,目录号:430186)
  28. 通用瓶(Thermo Fisher Scientific,目录号:BTS-402-050M)
  29. 温控震荡培养箱(例如Infors Multitron标准或Innova 4330)
  30. 温度控制培养箱(如 Panasonic,型号:MIR-254 PE或MIR-154 PE)
  31. 组织培养罩(例如Scanlaf Mars Safety 2)
  32. 超纯水供应(例如 Milli-Q ® Advantage A10超纯水净化系统)
  33. 高压灭菌器
  34. 水浴(例如 Grant SUB Aqua 12 Plus)\
  35. pH计和校准缓冲液(例如Hanna Instruments Edge Hybrid pH,目录号:HI-2020-02)
  36. 细胞计数器(例如Beckman Coulter Z sub 1)或血细胞计数器(Weber Scientific)
  37. 台式离心机(例如Thermo Fisher Scientific,型号:IEC CL30或Eppendorf,型号:5702R)
  38. 台式微型离心机(如 Eppendorf,型号:5424或5424R)
  39. 热循环仪(96×0.2ml管道模块)(例如Biometra Tdemement或Applied Biosystems Veriti)
  40. 可编程蠕动泵(例如 Watson Marlow,型号:505D i
  41. 8通道电子移液管(例如eLine50-1,200μl)(Sartorius,目录号:730391)和50-1200μl吸头(SafetySpace TM过滤嘴)(Sartorius,目录号码:79121F)
  42. 共聚焦显微镜(例如蔡司,型号:LSM710或LSM780)
  43. TIRF显微镜(例如尼康公司)
    注意:Zeiss共焦显微镜和尼康TIRF显微镜都随制造商的图像分析软件(分别为Zen和NIS-Elements)提供,但由于用户对此设备的时间有限,图像分析通常在专用的计算机工作站运行这些软件包是从制造商额外购买。免费使用的图像分析软件(开源)包也经常使用。 ImageJ和Fiji可用于共焦和TIRF图像。 ImageJ可以从 http://imagej.nih.gov/ij 和斐济 http://fiji.sc/Fiji
  44. 电穿孔仪(例如BIO-RAD Xcell)和1mm间隙宽度比色杯(Cell Projects,目录号:EP-201)
  45. Potter-Elvehjem homogensier(Wheaton,目录号:358049)
  46. 超速离心机(Beckman Coulter,型号:Optima MAX-XP)
  47. 轨道阱质谱仪(Thermo Fisher Scientific)
  48. 0.2ml具有12个孔和单独盖的PCR条带管(VWR International,目录号:53509-306)
  49. TLA-110转子(Beckman Coulter)
  50. XCell SureLock微型细胞和XCell II TM印迹模块(Life Technologies,Novex,目录号:EI0002)和电源(Bio-Rad Laboratories,PowerPac TM HC High - 目前电源,目录号:164-5052)

程序

  1. 生长盘基网柄细胞
    1. 如果可能的话,所有使用live 盘基网盘细胞应该是 在22℃的温度控制室中进行。 启动和 在振荡的悬浮液或在培养皿中的培养物的传代应该是   在组织培养罩中在无菌条件下进行。 这个很 重要的是每月从孢子或冷冻的股票更新股票 储存于-80℃或液氮中
    2. Ax2或TSPOON为空 细胞(HM1725和HM1727)细胞在摇动的悬浮液(22℃, 180rpm)在无菌培养基中在250ml或500ml锥形瓶中(种子 0.5-2×10 5个细胞/ml),并在中期对数期(2-5×10 6个细胞/ml)收获。在这些条件下的倍增时间通常为8-11小时。
    3. 或者,细胞可以在组织培养板上生长 收获时(每个平板应产生1-2×10 7个细胞) (图1)。当需要相对较少的共焦时,这是有用的  或TIRF显微镜

      图1.相位对比灯 变形虫的近融合草坪的显微照片。 细胞在10cm组织培养皿中生长并拍照 含有12ml无菌培养基。注意,之间有很少的间隙 个体细胞。刻度显示在右下角。

  2. 电穿孔盘基网织红细胞(基于Pang等人,1999)
    1. 收获中期对数期细胞到50ml无菌塑料管和 在300×g离心2-3分钟以沉淀细胞。 吸出 上清液并加入50ml冰冷的E50缓冲液并重悬细胞 轻轻敲击并摇动试管,轻轻擦拭
    2. 使用自动细胞计数器或血细胞计数器确定细胞数/ml
    3. 在300×g离心2-3分钟以沉淀细胞,然后 在冰冷的E50缓冲液中以4×10 7/sup/ml重悬。 放置单元格 悬浮在冰上并孵育5分钟(如果细胞开始沉降 然后通过搅拌轻轻混合)
      1. 对于过表达质粒 作为pJH101(TSPOON-GFP融合由组成型活性肌动蛋白驱动15  启动子)或pDT58和pDT61(均含有TSPOON-GFP融合驱动的  通过TSPOON启动子),将0.1ml细胞悬浮液转移至 预冷无菌电穿孔杯(1mm间隙宽度)并添加 10-30μg超螺旋质粒DNA(优选在≤15μl的Tris-HCl或  TE缓冲液中,并根据制造商的说明书制备 使用HiSpeed Plasmid Midi Kit),然后轻轻吹打混匀  以避免将气泡引入细胞悬浮液。返回 将比色皿放到冰上(图2)
      2. 最常见和可靠的基因 使用杀稻瘟素 S作为杀稻瘟素实现了 使用含有抗性盒的质粒(例如, 作为pLPBLP),其两侧均具有与靶标同源的DNA 基因(Faix等人,2004; Faix和Kimmel,2006)。这个 靶向/破坏盒应该从其余的 通过用限制酶完全消化来构建。挖空 TSPOON,用Apa I和Sac II切割质粒pDT70(基于pLPBLP) (这些酶在切割的DNA中产生3'突出端,但是酶 离开5'overahangs例如 BamH I或者可以使用平滑切割器,例如 Pvu 以任何组合释放破坏盒)。酶 其中通过单次酚萃取随后三次 用氯仿萃取,用乙醇沉淀的切出的DNA (Sambrook和Russel,2001)。以2-3mg/ml重悬浮切割的DNA Tris-HCl或TE缓冲液。添加15-20微克的破坏盒DNA 每个0.1ml细胞悬浮液,再次在冷冻无菌条件下 电穿孔杯(1mm间隙宽度)并如步骤B4a混合

        <图>图2中描述的电穿孔装置的照片 此协议。在后台,BioRad Xcell与pod一起显示 打开并装载电穿孔比色皿(1cm间隙宽度)。在里面 冰桶是两个等待电穿孔的小杯。 在里面 前列腺(15ml)含有将是的无菌培养基 加入(0.5ml),每个比色杯被脉冲两次以电穿孔 细胞悬液。 组织培养皿具有6个孔,其中每个   含有3ml的无菌培养基也显示在冰上回收后 5分钟,加入100μl电穿孔细胞悬浮液 到这个菜的每个井。

    4. 细胞/DNA悬浮液应当快速电穿孔以最小化比色杯中的细胞沉降。
      1. 从冰上取出比色杯,干燥任何多余的水分 所述电极在放入电穿孔舱之前具有组织。
      2. 参数应设置为0.75-0.85 kV,25μF电容   Bio-Rad Xcell的无穷大(∞)设置。 一样 参数在Bio-Rad GenePulser和GenePulserII上使用 它们可能需要用来自其他的电穿孔装置进行优化 制造商。
      3. 将两个脉冲输送到每个比色杯5秒 然后迅速加入0.5 ml无菌室温无菌培养基, 通过温和地吹吸上下混合,然后将比色皿放回冰中 5分钟。
      4. 对于筛选过程和筛选 转化体参见Faix等人(2004); Kimmel和Faix(2006); Muller-Taubeberger等人(2006)。

  3. 过表达和敲除细胞系的分离
    1. 对于每个比色皿准备在每个孔中具有3ml的无菌培养基的6孔组织培养板。
    2. 从冰中取出比色杯,轻轻地重悬细胞 移取,然后向组织培养皿的每个孔中加入0.1ml。
    3. 在22℃孵育过夜(通常16-24小时),然后加入, 重复孔,10μg/ml,20μg/ml和40μg/ml G418(对于质粒 pJH101和pDT61)。
    4. 对于用潮霉素选择,加入30μg/ml,60μg/ml和90μg/ml(对于pDT58)。
    5. 通过抽吸改变选择培养基,以去除死细胞   2-3天,并且孔在9-14天后应该汇合。 (潮霉素   似乎比G418杀死盘基网藻更有效   因此重要的是每2-3天更换培养基 旨在确保去除死亡和垂死的细胞将成为 从组织培养板分离)。
    6. 对孔进行筛选 通过上下吹打使细胞移动来优化表达 并取出0.3毫升到单室的有盖的盖玻片(8 好)。
    7. 使细胞沉降并附着20-30分钟 在每孔中的培养基被吸出并用0.3ml的 LOFLO介质或KK <2> C。
    8. 然后通过共焦或检查细胞 TIRF显微镜对GFP融合物的存在和分布 蛋白质(图3和4)。


      图3.TSPOON-GFP的过表达上图是用pDT61(TSPOON-GFP融合物)转染的细胞 由Tspoon启动子驱动的蛋白质)并选择14天 含有10μg/ml G418的无菌培养基。大多数细胞表达融合 蛋白质,但在不同水平。底部板是模拟转染的 细胞。将两个板中的细胞转移到LoFlo培养基中30分钟  分钟,然后通过共聚焦显微镜使用精确成像 相同的图像采集参数。注意微弱的自发荧光 模拟转染的细胞作为斑点可见。刻度表示 图的右下角。


      图4. TIRF图像 TSPOON-GFP。如图3所示转染和选择变形虫。 将细胞转移到KK 2 C(加50μg/ml L-抗坏血酸)3小时 在图像收集之前。 显示刻度。

    9. 做一个   永久性原液,收获具有最佳表达的孔 通过向上和向下移液无菌介质以移除细胞 然后用于启动更大的培养物(用适当的培养基生长) 水平的G418或潮霉素)在摇动悬浮液或几十 cm组织培养皿,每个具有12ml的无菌培养基。
    10. 一个 最少8×10 7个细胞应该被收获和沉淀 离心(300×g离心2分钟),然后重悬于1.6ml水中 冷冻介质并如步骤C23中那样处理
    11. 分离 敲除突变体应将细胞悬浮液稀释至适当 密度以确保在细胞存活范围内的克隆性。 这是 当靶基因影响生长时特别重要,因为 野生型非同源重组体可能然后长出这样的 剔除,使屏幕及其隔离更加困难
    12. 至   确保克隆性,细胞悬浮液从单个比色皿是第一 加入到总计15ml的无菌介质中,在50ml塑料管中   细胞为约2.67×10 5个/ml。
    13. 这个单元格 悬浮液进一步稀释10(2.67×10 4个/ml),50(5.33×10 3个/ml) 100(2.67×10 3个/ml),200个(1.33×10 3个/ml)和400倍(0.67×10 3个/)。 每个稀释物制成总共15ml的无菌培养基和100 μl/孔分配到单独的96孔组织培养板中 每次稀释
    14. 通常制备4×96孔板 100,200和400倍稀释,因此50ml稀释的细胞应该是 为这些稀释液中的每一种制备。
    15. 孵育在a 潮湿的气氛(如在一个大塑料蛋糕储物盒内衬   湿组织)在22℃过夜(通常16-24小时),然后加入100μl   的含有20μg/ml杀稻瘟菌素S的无菌培养基 μg/ml终浓度)。
    16. 更改选择培养基以去除死细胞 每2-3天通过有力地"投掷"介质到大组织 衬里(停止溅回井)容器。加入200μl新鲜 含有10μg/ml杀稻瘟菌素S的无菌培养基。
      注意:将细胞放入96孔板中并补充  介质最好用配备8通道电子移液器  提示每个容量为1,200μl。细胞悬浮液或新鲜 介质从无菌塑料容器中分配。这些水库 可以在steribag小袋中高压灭菌后重复使用。
    17. 96 包含最少稀释的细胞的孔板应该具有汇合 孔,而含有100,200和400的板 倍稀释可能需要长达21天(基于的平均倍数 使用许多不同的敲除载体,但缺失基因 损害无菌培养基中的生长可能增加这些时间)。 通常从其中<30孔/板的板中筛选稀释物 占据,因为这些可能是克隆的。
    18. 每个细胞   通过吸取上下收集汇合的孔,然后转移   到含有0.75ml无菌KK 2 Sub的1.5ml微量离心管中(这 是在细胞沉淀后稀释残留的无菌介质)
    19. 补充井200μL的新鲜无菌介质含有10 μg/ml杀稻瘟菌素S.将细胞在台式微量离心机中沉淀 全速20秒,并通过抽吸除去上清液。
    20. 然后可以使用a。分离来自细胞沉淀的基因组DNA 各种商业试剂盒,但我们更喜欢Quick-gDNA MiniPrep试剂盒 从Zymo Reasearch,因为它是快速,有几个步骤和可重复 产生良好质量的基因组DNA,用于使用各种PCR进行筛选 聚合酶[Expand 20 kb plus polymerase(Roche)] 最适合TSPOON屏幕,但我们也使用了Q5,Pwo和KOD 聚合酶]。
    21. 在TSPOON敲除的情况下,隔离和   筛选从选择开始直到汇合17天 其中在含有200和400倍的平板中观察到 稀释。
    22. PCR筛选使用TSPOON特异性引物(TCP)寡核苷酸TCP15:(5'-GATGAAATTTATCAGATATTGATTTCATGAATGTTTCACC-3')和TCP16(5'-CTGCTGATGTTGGTTTATAGGTGGCAAACCACCATC-3' 中断盒以最小化由单个产生的假阳性 交叉重组事件。 编码TSPOON( tstD , DDB_G0350235)位于染色体1上并且侧接基因 DDB_G0271002和DDB_G026990。 TCP15的确切基因组位置是 1:3986383..3986422和TCP16是1:3982321..3982356。 一切 本方案中使用的寡核苷酸引物位于这些序列中 坐标。
      1. PCR反应(在0.2ml管中)由a 总共25μl,含有1×Expand 20kb PLUS缓冲液,各0.4nM 引物,5μlQuick-gDNA小量制备物,500μMdNTP和0.25μl(5μl) 单位)Expand 20kb PLUS 聚合酶
      2. 循环参数为   步骤(1)92℃1分15秒; (2)92℃30秒; (3)52℃45分钟   秒; (4)62℃7分钟; (5)62℃,5分钟。 步骤2到4 重复另外36次,然后将反应在4℃下保持直至   通过凝胶电泳分析(Sambrook和Russel,2001)
      3. 来自野生型克隆的PCR产物是〜4.1kb,TSPOON敲除克隆〜4.7kb。
      4. 杀稻瘟素抗性盒和侧翼DNA的插入 将限制性位点引入敲除PCR产物 缺少野生型。 这是一个有用的进一步证明正确 靶向作为用限制酶消化这些产物 作为TSPOON的SmaI,留下野生型产物完整,但切割 敲除产物分成3个片段。
      5. 共有46口井 通过该方法筛选,37个是TSPOON敲除,6个是野生型,3个   不是克隆的,因为它们含有两种PCR产物。 因此, 该基因的靶向频率为80%,尽管高的是 与我们实验室中该程序靶向的其他基因保持一致   例如对于 iplA ,67% mscS ,57% mclN ,50% phdA 和12% em> dagA 。
    23. 从不同的96孔板分离的四个TSPOON敲除物进一步处理(2个用于研究,2个作为备份)。
      1. 如前所述收获含有这些细胞的孔,即细胞   以KK 2稀释至0.6-1.2个细胞/μl(汇合的孔含有〜10个细胞)。
      2. 制备SM琼脂平板,每个具有0.4ml滴致密产生的产气荚膜杆菌细菌悬浮液(图5)。


        图5.覆盖在产气杆菌属的草坪中的SM琼脂板,其具有可见的盘基网柄菌群集落。 菌落由营养性阿米巴组成,而在中心结果 体和其他多细胞结构已经形成。殖民地适合  收获和冷冻应为〜1至1.5厘米的直径和 明显与邻居分离。在该实施例中合适的菌落是  由黑色箭头指示。在10微升的大约6个阿米巴 KK 2直接加入400μl滴在产板上的致密克雷伯菌(Klebsiella aerogenes)的悬浮液,然后均匀铺展。 平板后4天可见集落,并拍摄该照片  6天后。刻度显示在右下角。

      3. 添加10微升的稀释细胞悬液到此滴,并用无菌塑料撒布均匀地铺展在板上。
      4. 离散菌落在4-5天内出现,并且当它们已经达到〜1.5cm直径时收获一个。
      5. 使用无菌小金属刮刀的平端 将整个菌落直接转移到1.6ml冷冻培养基中 在冰上的CryoTube。 间歇地涡旋冷冻管 确保均匀悬浮,然后转移至冷冻保存 模块冷却至4℃。
      6.  然后将模块传送到 -80℃冷冻过夜,然后将管保持在-80℃(细胞 保持活性> 2年)或转移到液氮中 储罐(不确定的活力)。

  4. 显微镜
    1. 固定和活细胞分析我的显微镜在营养或聚集的发展阶段。

      对于活植物细胞
      1. 只是收获生长细胞从无性文化和转移 适当数量的细胞,使得最终密度为0.5-1.5×10 5/cm 2/cm 2至玻璃底盘或含有LOFLO培养基的带盖的盖玻片 (这已经被制定以最小化背景和细胞 自体荧光相比标准的无菌介质,它会保持   细胞营养一段时间)或KK 2 C(它们将开始发育 这种缓冲液,但是自发荧光和光敏性将是 最小化)。
      2. 在22℃孵育10分钟,以允许细胞 然后吸出培养基(不要让细胞干燥)和 更换为新鲜的LOFLO或KK <2> C。 它们然后可以用于活细胞 成像或固定。

      对于开发的细胞
      1. 将0.5-1.5×10 5/cm 2/cm 2 转移到KK <2> C中,并且一旦将KK < 细胞已附着。
      2. 在22°C孵育8-10小时,以允许细胞成为聚集能力。
      3. 或者,将1×10 6个细胞转移到35mm组织培养物中 含有2ml KK sub 2 C的培养皿,允许细胞附着然后更换 KK sub 2 C并在22℃孵育1小时。
      4. 转盘到 15℃过夜(15-17小时),然后将培养皿返回至22℃1小时 在转移前(0.5-1.5×10 5/cm 2/cm 2)向玻璃底盘(细胞  应在22℃下在2-3小时内聚集)。
      5. 如果数量较大 的开发的细胞,然后在2x重悬营养细胞 10 /ml,在KK 2 C中,并将其转移到具有体积 至少为细胞悬浮液的5倍以确保最佳通气。  将细胞在180rpm下在22℃下振荡1小时,然后是100μl小滴 的KK sub 2 C,含有足够的cAMP(从主原料稀释) 整个悬浮液的终浓度为100nM,分配  进入悬浮液每6分钟3.5-5.0小时通过可编程 蠕动泵。这个程序模拟cAMP的脉动波  通过发展中的变形虫群体发射并且是有用的 对于在早期发育和cAMP信号传导中有缺陷的突变株
      6. 收获细胞并通过离心300×g离心沉淀2分钟, 吸出上清液并以1×10 7 /孔/ml重悬细胞 KK 2 C和如前所述的板在0.5-1.5×10 5/sup/cm 2 。
      7. 有 用于免疫细胞化学(ICC)的多种方法修复盘基网藻   并且它们可以在别处找到(DictyBase1和2,Jungbluth等人 1994; Hagedorn等人,2006)。
    2. 共聚焦激光扫描显微镜。
      1. 活细胞成像通常是检测标记之间的平衡  融合蛋白(信噪比)和损伤细胞 激光由于与照射相互作用产生的自由基  细胞成分或荧光团标签。使用10x或20x镜头 这从来不是一个问题,但在这些放大倍数 在细胞内几乎没有或没有空间分辨率,因为盘基网柄细胞直径为〜10-15μm。
      2. 通常为63x或  需要100x透镜来解析标记的亚细胞定位  蛋白质在盘基网藻中。
      3. 植物性变形虫 特别是对来自较短波长的损伤特别敏感 例如405nm激光。大多数配备氩激光器(25-35 mW) 共聚焦显微镜用于成像YFP,GFP和CFP融合蛋白 过度的功率会导致光毒性和光漂白 激光强度应保持最小(对于488通常为2-10%) nm激光线)。
      4. 更长波长的激光线,如561 mn 用于RFP标记的蛋白质存在较少的问题并且可以使用 在较高功率(> 15%)的信号可以通过打开来增加 针孔
      5. 避免线平均,慢扫描和实时反卷积。
      6. 发达的细胞通常对激光损伤较不敏感,但它 可以是融合蛋白依赖性。 例如GFP标记的蛋白质 驻留在质膜中可以使细胞更敏感(由于 到膜中的脂质氧化)比胞质蛋白。 可以加入L-抗坏血酸(最终50-100μm)以清除自由基   以尽量减少这个问题和维生素E类似Trolox C也 用于此目的(向生长培养基中加入1mM并孵育 成像前过夜)。
      7. 如果光毒性不能克服 然后考虑切换到旋转盘共聚焦显微镜(SDCM) 其中照明限于数千个小共焦体积 而不是通过整个样本 减少光漂白和光毒性。
      8. 跟着 应每2-30秒捕获营养细胞框架的运动 和用于发育的细胞每1-20秒。 最快的现实帧速率   是1/sec。 可能需要减少从扫描的区域 默认512像素×512像素到512×300,因为这允许较慢的扫描   (更好的图像)并且最大化帧速率(Frigault等人,2009; Muller-Taubenberger和Ishikawa-Ankerhold,2011)。
      9.  用于单元格 表达启动子TSPOON-GFP构建体使用的典型设置 488nm激光功率设置在5%,针孔打开到2.5艾里 每秒收集512像素×300像素帧的单位。 对于 更快的时间分辨率切换到SDCM。
      10.  使用固定单元格 和ICC光漂白可能是一个问题,但使用抗褪色 安装介质(通常为Fluoromount-G ®)可以最小化这一点并允许 较慢的扫描速度和平均值,以提供更好的图像。
    3. TIRF显微镜。
      1. 光毒性的问题通常与营养的盘基网藻细胞相比更加尖锐,部分是因为尽管瞬逝场是 限于来自盖玻片的样品的初始〜100nm 质膜被强烈照射。
      2. 已在LOFLO或KK 2 C的图像单元至少一小时。 可能需要包含L-抗坏血酸。
      3. 保持激光功率和照明时间最小。
      4. 由于没有扫描,所以帧速率更高。 100x镜头 (高NA)应与附加的1.5倍变焦镜头一起使用,如果存在   这允许ameobae被非常详细地成像。
      5. 过滤所有缓冲液(0.22μm)以去除颗粒物。
      6. 表达启动子_TSPOON-GFP的细胞的典型设置 构造具有设置为7%的488nm激光功率,曝光时间为 0.08秒,以高达12每秒收集512×512帧。

  5. 细胞分级
    1. 培养表达A15_GFP(对照)或启动子-TSPOON-GFP的细胞 直到它们在选择性培养基中达到2-4×10 6个/ml的密度, 显微镜检查> 50%的细胞表达GFP。
    2. 从a开始   最大8×10 8个细胞,将细胞在KK 2缓冲液中洗涤,然后 在300×g下沉淀3分钟。 所有后续步骤在4℃进行 C。
    3. 将细胞重悬于具有蛋白酶抑制剂的PBS中 鸡尾酒,通过电动Potter-Elvehjem匀浆器的8次冲击裂解 (研磨室间隙0.1-0.15mm),然后通过a进行5次冲程   21-g针以确保完全裂解。
    4. 或者,细胞可以 以1-5×10 8/sup/ml重悬,并置于装有25℃的注射器中 mm过滤器保持器包含预过滤器和Nucleopore过滤器(3.0 μm)。 然后使细胞悬浮液通过该过滤器组件 两次。
    5. 然后将裂解物在4,100×g离心32分钟   沉淀核和未破裂的细胞,以及核后上清液 进一步在50,000rpm(135,700×g RCF最大)下离心30分钟,在 TLA-110转子,以回收膜沉淀和胞质上清液。
    6. 使用标准蛋白质测定来评估2个级分中的蛋白质回收率,并调节相等蛋白质的体积。
    7. 或者,为了平衡体积,胞质溶胶上清液 通过用10%三氯乙酸在4℃下沉淀来浓缩   30分钟,并通过14,000×g离心10分钟回收。 样品 用三氯乙酸沉淀,用冰冷的丙酮洗涤   (-20℃),空气干燥2分钟,然后重悬于相同体积中 作为沉淀级分。

  6. 蛋白质下拉
    使用在组成型(A15_TSPOON-GFP)和其自身启动子(prom_TSPOON-GFP)下稳定表达TSPOON-GFP的变形体进行免疫沉淀,并且将未转化的细胞用作对照。此处给出的量推荐用于大规模免疫沉淀,足以在天然条件下进行相互作用蛋白的蛋白质组鉴定。起始培养物的大小和所有随后的体积可相应地减小以进行较小规模的免疫沉淀,例如其中蛋白质的鉴定通过蛋白质印迹法进行。该协议也可以修改为变性(通过加热和/或SDS的存在但在此不详述)免疫沉淀。
    1. 使细胞生长直到它们达到2-4×10 6 /μL/ml的密度 选择性培养基,和通过显微镜检查> 50%的细胞表达GFP。
    2. 通过在300×g离心2分钟使高达8×10 8个细胞沉淀,在50ml的KK sub缓冲液中洗涤两次,然后重悬 以2×10 7个细胞/ml在KK 2缓冲液中稀释,并在22℃下饥饿4-6小时,同时 以180rpm振摇。
    3. 然后将细胞在300×g下沉淀3小时  min,在4ml PBS-T加蛋白酶抑制剂混合物片剂中裂解, 在4℃下在15ml管中以8rpm旋转提取20分钟,和  然后旋转20,000×g,15分钟以除去碎片和不溶物 材料。
    4. 通过BCA蛋白测定,得到的裂解物含有10-15mg总蛋白。
    5. 通过加入100μlPA琼脂糖珠将裂解物预澄清 浆液(50%v/v的PBS溶液),并以8rpm旋转温育30分钟,   随后在2200×g离心3分钟以沉淀珠粒。
    6. 将上清液转移(〜5ml)至新管中, 是免疫沉淀(蛋白质)的起始材料 浓度应为2-4mg/ml)。
    7. 为最大回复 裂解物使用针对GFP的内部抗体进行免疫沉淀 在4℃下旋转过夜,尽管孵育可能是足够的 只需90分钟。
    8. 适当的抗体浓度   需要单独优化,尽管推荐的起点   通常比用于Western印迹的高2至5倍。
    9. <与抗GFP孵育后,将50μlPA琼脂糖珠浆液(50%   v/v的PBS溶液)在4℃下以8rpm旋转加入90分钟。
    10. 一种替代方法是使用多克隆的商业来源 抗GFP或已经偶联到琼脂糖的抗GFP 例如具有抗体保持偶联的益处的GFP-TRAP 当免疫沉淀的蛋白质从中洗脱时,与PA琼脂糖结合   珠。 在这种情况下,在该步骤中加入PA琼脂糖 省略。
    11. 通过离心回收抗体复合物 2200×g 3分钟,然后用10ml PBS-T洗涤两次,重悬浮   在1ml PBS中,并转移到1.5ml微量离心管中。
    12. 用PBS洗涤抗体复合物两次, 在8,000×g下将珠粒沉淀20秒,然后从中洗脱 珠用200μl洗脱缓冲液温热至60℃10分钟。
    13. 然后将珠子以8,000×g沉淀20秒,并取上清液   小心移除; 这可以通过使用细的凝胶载荷来促进 提示删除最后50微升作为珠子不进入提示,或通过 使用微生物自旋色谱柱。
    14. 洗脱蛋白   然后在-20℃下用1.2ml丙酮沉淀过夜(3-20小时) 并在10,000×g离心5分钟,4℃。 过夜 沉淀增加产量,特别是对于低蛋白 浓度,但延伸超过这个结果导致较低的恢复
    15. 除去上清液,将沉淀物空气干燥2分钟 重悬于选择的缓冲液中(例如 1x LDS样品缓冲液) SDS-PAGE)。
    16. 对于样品的SDS印迹,使用SDS-PAGE凝胶 运行并根据标准方案转移(Sambrook和 Russel,2001)。
    17. 对于蛋白质组学,样品在预制(pre-cast)上运行 NuPAGE 4-12%Bis-Tris凝胶,用考马斯G-250 SimplyBlue染色 SafeStain,然后切成8个凝胶切片。 处理每个凝胶切片 通过过滤辅助样品制备溶液消化,并且样品 通过液相色谱 - 串联质谱分析 轨道阱质谱仪。
    18. 下来的蛋白质 非转化对照,以及任何具有较少的蛋白质 多于5个已鉴定的肽,不一致的蛋白质 coimmunoprecipitate在三个独立的实验,或蛋白质 与诱饵相比非常低的丰度(即,摩尔比) <0.002)。 认为其余的蛋白质是特异性的 免疫沉淀。

食谱

  1. 轴介质
    35.5g HL5用葡萄糖
    200mg二氢链霉素 注意:不要使用链霉素,因为它在高压灭菌过程中失活。
    将超纯的dH 2 O加入到1,000ml
    中 分配到锥形瓶
    75毫升/250毫升烧瓶用50毫米×38毫米泡沫塑料塞子塞住 150毫升/500毫升烧瓶用50毫米×38毫米的泡沫桶
    塞住 750毫升/2000毫升烧瓶用50毫米×50毫米泡沫塑料塞子塞住 每个烧瓶的顶部应覆盖一小片(12厘米×12厘米)铝箔,以防止塞子的顶部在存储期间被灰尘污染
    高压灭菌
    注意:121℃不超过15分钟,并尽快从高压釜中取出以避免焦化,即介质变成深褐色。使用更大的更复杂的高压釜,具有延长周期时间的额外安全功能,这可能需要降低到7分钟。
    或者过滤灭菌(Steritop 1000 ml单位,0.22μm)到无菌1,000 ml Duran瓶中,或使用1,000 ml Corning过滤器单元(0.22μm)
    储存在黑暗中22°C
  2. LOFLO介质
    8.4克LOFLO培养基
    100mg二氢链霉素 将超纯的dH 2 O加到500ml
    中 使用500ml Stericup过滤器单元过滤灭菌(0.22μm) 在22°C(短期)或4°C(长期)存放在暗处
  3. SM琼脂
    41.7g SM琼脂
    将超纯的dH 2 O加入到1,000ml
    中 高压灭菌
    注意:121°C不超过15分钟,并尽快从高压釜中取出,以避免焦糖化。使用更大更复杂的高压釜,具有延长周期时间的额外安全功能,这可能需要减少到7分钟。
    在加热的水浴中冷却至60℃,然后倾倒25-30ml/10cm直径的塑料培养皿
    一旦琼脂凝固并冷却,将容器放在密封的盒子中,或者在塑料袋(使用包装好的袋子)中分批封装(15-20个板)以使干燥最小化
    在22℃(短期<1周)或4℃(长期)的黑暗中贮存
  4. SM肉汤
    24.7g SM肉汤
    将超纯的dH 2 O加入到1,000ml
    中 向每个20ml通用瓶中分配10ml。
    高压灭菌
    注意:121°C不超过15分钟,并尽快从高压釜中取出,以避免焦糖化。 使用更大的更复杂的高压釜,具有延长周期时间的额外安全功能,这可能需要减少到低至7分钟。
    储存在黑暗中22°C
  5. KK 2 缓冲
    2.24g KH 2 PO 4 4/
    0.52g K 2 HPO 4,无水
    将超纯的dH 2 O加入到1,000ml
    中 高压灭菌器在1,000 ml Duran瓶中灭菌(121°C,15分钟)
    高压灭菌后的pH应为〜6.1
    加入2ml无菌1M MgSO 4 储存于22°C
  6. KK 2 C buffer
    1,000ml无菌KK 2
    0.1ml无菌的1M CaCl 2·h/v 储存于22°C
  7. 1 M MgSO 4/v/v 246.48g MgSO 4 将超纯的dH 2 O加入到1,000ml
    中 高压灭菌器在150ml Duran瓶(121℃,15分钟)中灭菌
    储存于22°C
  8. 1 M CaCl 2
    147.02g CaCl 2
    将超纯的dH 2 O加入到1,000ml
    中 高压灭菌器在150ml Duran瓶(121℃,15分钟)中灭菌
    储存于22°C
  9. 浓缩的克雷伯菌(Klebsiella aerogenes)
    准备带有克雷伯菌(Klebsiella)的SM琼脂板,以便有单个菌落(储存在8℃,每2个月从冷冻储存液更新)
    用火焰灭菌的铂环(或一次性无菌塑料针或塑料环)挑取菌落,并使用该菌种接种含有SM肉汤的通用瓶子
    在22°C下孵育瓶子48小时,然后使用
    进行涡旋 储存在黑暗中8°C
    每月续订
  10. 电穿孔缓冲液E50
    2.38克HEPES
    1.86克KCl
    0.29g NaCl
    0.5ml的1M MgSO 4 0.21g NaHCO 3水溶液 0.08g NaH 2 PO 4 subO 2·2H 2 O O 将超纯的dH 2 O加到500ml
    中 用KOH调节至pH7.0
    使用500ml Stericup过滤器单元进行过滤灭菌(0.22μm),并在4℃下贮存
  11. Tris-HCl缓冲液
    0.12 g Trizma ® base
    将超纯的dH 2 O加至100毫升
    用HCl
    调节至pH 8.0 高压灭菌器在150 ml Duran瓶中灭菌
    储存于22°C
  12. TE缓冲区
    0.12 g Trizma ® base
    0.04g EDTA
    将超纯的dH 2 O加至100毫升
    用HCl
    调节至pH 8.0 高压灭菌器在150 ml Duran瓶中灭菌
    储存于22°C
  13. 20mg/ml G418
    将552mg遗传霉素G418硫酸盐调整至724μg/mg的效力
    注意:效力因批次而异。
    将超纯的dH 2 O加至20ml
    用装有0.22μm注射器过滤器的20ml注射器过滤灭菌 在-20℃下以1ml等分试样保存在无菌1.5ml微量离心管中,G418溶液保持其效力> 2年。 工作库存储在4℃<6℃,并在6个月内使用
  14. 冻结介质
    150毫升马血清
    11.25ml [7.5%(v/v)] DMSO 使用150ml Stericup过滤器单元过滤灭菌(0.22μm) 分装入无菌的50ml管中,储存于-20°C,应在1年内使用
  15. 50mM L-抗坏血酸 0.22g L-抗坏血酸 将超纯的dH 2 O加至25ml
    用装有0.22μm注射器过滤器的50ml注射器过滤以除去微粒
    以0.5ml等分试样在-20℃下保存
  16. cAMP的主证书
    6.6克cAMP
    将超纯的dH 2 O加至95ml
    用KOH非常小心地调节到pH7.0(注意:所有cAMP在pH <5时不溶解)
    加入超纯的dH 2 O至最终体积为100ml
    使用150ml Stericup过滤器单元进行过滤灭菌(0.22μm),并以-20ml等分试样储存在-20℃下。
  17. PBS
    8.01g NaCl
    0.20克KCl
    1.42g Na 2 HPO 4水溶液,
    0.24g KH 2 PO 4 sub/
    中 在500ml Duran瓶中高压灭菌(121℃,15分钟)
    储存于22°C
  18. PBS-T
    PBS,用10%Triton储液
    调节至1%Triton X-100 过滤灭菌长期储存(0.22μm)
    储存在22°C或4°C(长期储存)
  19. 三氯乙酸
    100克三氯乙酸 溶解在35ml超纯的dH 2 O中 然后用超纯的dH 2 O *补充至100ml 储存于22°C
  20. 洗脱缓冲液
    1.21 g Trizma ® base
    将超纯的dH 2 O加至75ml
    20ml来自10%溶液的2%(w/v)SDS溶液 用HCl
    调节至pH 8.0 加入超纯的dH 2 O至最终体积为100ml
    使用150ml Stericup过滤器单元进行过滤灭菌(0.22μm),并以1ml等分试样以
    -20°C

致谢

本研究由医学研究委员会[(MC_U105115237),DT和RRK]和The Wellcome Trust(JH)支持。盘基网柄菌的电穿孔是基于Pang,Lynes和Knecht的方法 (Pang等人,1999)。

参考文献

  1. Bloomfield,G.,Tanaka,Y.,Skelton,J.,Ivens,A.and Kay,R.R。(2008)。 实验室股票基因组广泛重复 基因组生物 9(4):R75。
  2. DictyBase 1 http://www.dictybase.org/techniques/geneex/indirect_immunofluo.html。
  3. DictyBase 2 http://www.dictybase.org/techniques/microscopy/fixation_rgomer.html。
  4. Faix,J.,Kreppel,L.,Shaulsky,G.,Schleicher,M。和Kimmel,A.R。(2004)。 使用快速有效的方法在迪氏盘基网柄菌中产生多种基因破坏单个可选择标记和Cre-loxP系统。 核酸研究 32(19):e143。
  5. Hagedorn,M.,Neuhaus,E.M。和Soldati,T。(2006)。 优化了盘基网柄细胞的固定和免疫荧光染色方法。 Methods Mol Biol 346:327-338
  6. Hirst,J.,Schlacht,A.,Norcott,J.P.,Traynor,D.,Bloomfield,G.,Antrobus,R.,Kay,R.R.,Dacks,J.B.and Robinson,M.S.(2014)。 TSET的表征,一种古老而广泛的膜贩运综合症。 Elife 3:e02866。
  7. Jungbluth,A.,von Arnim,V.,Biegelmann,E.,Humbel,B.,Schweiger,A.and Gerisch,G。(1994)。 抑制氧化磷酸化后肌动蛋白酪氨酸磷酸化的强烈增加:与肌动蛋白中的可逆重排相关骨髓的 Dictyostelium 细胞。 107(Pt 1):117-125。
  8. Kimmel,A.R.and Faix,J。(2006)。 使用Cre-loxP系统产生多个敲除突变体。 方法Biol 346:187-199.
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Copyright Hirst et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Hirst, J., Kay, R. R. and Traynor, D. (2015). Dictyostelium Cultivation, Transfection, Microscopy and Fractionation. Bio-protocol 5(11): e1485. DOI: 10.21769/BioProtoc.1485.
  2. Hirst, J., Schlacht, A., Norcott, J. P., Traynor, D., Bloomfield, G., Antrobus, R., Kay, R. R., Dacks, J. B. and Robinson, M. S. (2014). Characterization of TSET, an ancient and widespread membrane trafficking complex. Elife 3: e02866.
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