pCL-Ampho

逆病毒包装载体pCL-Ampho的使用方法——逆病毒包装与转染方法



293T Growth Medium:

   450 ml DMEM (JRH Biosciences)
   50 ml FBS (JRH Sciences)
   5 ml Glutamine (200 mM in saline, JRH Sciences)
   2.5 ml Gentamycin (10 mg/ml in water, Sigma)

HC11 Growth Medium:

  5 ml L-Glutamine (200 mM in saline, JRH Sciences)
  2.5 ml Bovine Insulin (1 mg/ml in 0.01 N HCL, Sigma)
  2.5 ml Gentamycin (10 mg/ml in water, Sigma)
  0.5 ml EGF (10 μg/ml in water, Gibco)
  50 ml FBS (JRH Sciences)
  440 ml RPMI (JRH Sciences)


Day 0: Split 293T cells for transfection:

  Wash cells off plate with 10 ml Hanks and put into 15 ml Falcon tube
  Add 1 ml 10X trypsin and mix by inversion for about 1-2 minutes
  Spin at 1000 rpm in clinical centrifuge for 1-2 minutes
  Resuspend cells in 10 ml growth medium
  Count cells
  Plate cells at 1X106 cells per 100 mm dish


Day 2: Transfect 293T cells:

  Plan transfection using 2 μg total DNA and 12 μl FuGene in 200 μl total 
  volume per 100 mm dish
  Dilute Fugene: 12 μl per reaction in serum free DMEM for total volume of
  200 μl

  Mix DNA plasmids in polypropelene tubes 

(Falcon #2063):
     1 μg DNA of interest
     1 μg pCL-Ampho
     Add 200 μl diluted FuGene dropwise to each DNA tube

  Incubate RT 15 min
  Feed cells with 5 ml fresh growth media and add FuGene/DNA mixture to media
 

Day 3: Change medium:

  • Remove viral medium and CAREFULLY feed with 6 ml growth medium
  This step may reduce a cytostatic factor (produced after transfections) that
  can inhibit growth and infection of your target cells.

  Split HC11 cells for infection
  Seed HC11 cells at 300,000 cells per 100 mm dish
  If using coverslips, incubate with FBS for at least 1 hour prior to plating


Day 4: Infect target (HC11) cells:

  • Collect virus-containing media off 293T cells
  • Syringe filter media through .45 μm filter onto target cells
   (Evaporation O/N and loss in filter leaves about 5 ml per plate)
  • Add 10 μl 5mg/ml polybrene (final conc = 10 μg polybrene/ml media)
   (Store polybrene in aliquots to decrease freeze/thaw cycles)
  • Wrap plates in parafilm
  • Spin cells in clinical centrifuge 10 min at 1800 rpm
  • Rotate plate 1/3. Spin 10 min at 1800 rpm
  • Rotate plate 1/3. Spin 10 min at 1800 rpm
  • Remove viral media and replace with fresh growth media


Day 6+: Harvest HC11 cells:

  • Wash cells with HBSS
  • Scrape remaining cells, pellet and flash freeze for protein/RNA analysis

  Stain cells for beta-gal expression:

  Fixing Solution :
    1.35 ml 37% Formaldehyde
    0.2 ml Gluteraldehyde
    1X PBS to 25 mls

  Staining Solution:
    625μl 40 mg/ml X-gal in DMF
    50 μl 1M MgCL2
    750 μl 100 mM Potassium Ferricyanide
    750 μl 100 mM Potassium Ferrocyanide
    1X PBS to 25 mls
    [100 mM Potassium Ferrocyanide = 2.11 g/50ml PBS]
    [100 mM Potassium Ferricyanide = 1.64 g/50 ml PBS]

  Store at room temperature, protect from light.
    1. Make -gal solution and warm in 37°C water bath to prevent crystallization
    2. Aspirate off media
    3. Wash cells 1X PBS
    4. Fix cells 5 min at RT in fixing solution
    5. Wash 3X PBS
    6 Optional: Add PBS to cells and warm plate at 37°C 5 min (reduces
      crystallization)
    7. Stain cells with staining solution at 37°C for 1-24 hrs
    8. Store stained cells in 70% EtOH at 4°C
      (EMBO J. 5: pg 3133, 1986)


Note:

   HC11 and MEC primary culture cells are very adherent and can withstand
   the force of spinning the plates at 1800 rpm. We found that HC11 cells died at
   2200 rpm. However, this is cell type-dependent, and spinning speed should be
   optimized accordingly.
   Expect to see -gal staining at the periphery of the plate. During the spin
   infection the media is pushed to the outside. The cells in the center of the plate
   will not be exposed to virus and will not be efficiently infected with retrovirus.