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Blastocyst Injection
发布时间:2019-01-20 12:00:00来源:深圳市第二人民医院 分享:

  

  Embryo preparation: 

  E2.5 culture in M16 for 1 day or 2 days to blastocyst 

  ES cells preparation: 

  ES cells for injection should be in the log phase of growth. Es cells that are nearing their maximum colony size and are reaching cofluency, or those exhibiting overt and extensive differentiation, generally do not produce good levels os chimerism. 

  

  1. 35mmor 60mm ES cells dish
  2. Passaged 1:2 the day before injection or change fresh medium 3~4hrs before injection
  3. wash twice with D-hank’s
  4. Trypsinize with 1 ml 0.25% Trypsin+0.04% EDTA
  5. Add 4 ml ES-medium to stop reaction
  6. Pipette into single cells
  7. Plate 1/4~1/2 suspension onto 0.1% gelatin coated dish
  8. 37°C, 5% CO2, 30 min~1 hr (During this time, the feeder cells will attach)(1hr or longer may be better for the feeder cell can attach the dish harder, yet the ESC still can be blown off from the feeder cell, with the result there is letter feeder cell in cell medium)
  9. Remove the medium and discard all floating ES cells
  10. Add 5 ml ES-medium, gently blow off the loosely adhering ES cells(10ml peptide blow the cells with lowest speed, we getting better result for more ESC, very little feeder cells in the cell medium)
  11. 1000 rpm, 3 min
  12. resuspend in 1 ml ES-medium or injection medium
  13. store 4°C for use (usable in 3~4 hrs)

  Injection medium preparation: 

  Injection medium: 

  500 ul ES-medium 

  10 ul 1M HEPES (in MilliQ, filter to sterilize) 

  + 2 IU DNase I/100 ul injection medium (add before use) 

  store 4°C 

  Injection: 

  

  1. Prepare injection chamber, 4°C
  2. Transfer 10 blastcysts and 3~5 ul ES cells to the injection chamber
  3. 4°C
  4. Prepare injection needle and holder
  5. 10~15 ES cells/ blastocyst
  6. Culture in ES-medium ~3 hrs for injected blastocysts to reexpand
  7. Uterine Transfer to 2.5-day pseudopregnant recipients. 6~8 embryos per side.
  8. 17 days later the pups will be born

  DNase I : 

  DNase I dilution buffer (10ml):                final conc. 

  1M Tris.HCl (PH 7.5)       50ul         10mM 

  NaCl                  0.0438g        150mM 

  25mM MgCl2              200ul          1mM 

  MilliQ                    4.7ml          

  Glycerol                   5 ml           50% 

  Dissolve 2mg DNase I in 2 ml DNase I dilution buffer 

  Mix by gentle inversion 

  Aliquot and store -20°C