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Protocols

Preparation of ES Cells for Blastocyst Injection

Five days prior to injection

  1. Thaw a vial of ES cells in a 37°C water bath
  2. Transfer contents of the vial into a conical tube with 10 ml ES cell media to dilute the freezing medium and also to disperse the cells
  3. Pellet the cells by slow centrifugation (< 1500 rpm) for 5 minutes
  4. Remove the supernatant and tap the tube gently to disperse cells
  5. Add 4 ml ES cell media and transfer to a fresh 6-cm feeder plate
  6. Culture in tissue culture incubator (37°C, 5% CO2) for 2 to 3 days, changing media daily
  7. Passage the cells by plating 500,000 cells onto a fresh 6-cm feeder plate
  8. Change media daily
  9. ES cells are best injected two days after passaging

 

Day of injection

  1. Refeed the culture the morning of injection
  2. Refeed the culture again 2 hours prior to injection
  3. Prepare 25 ml of blastocyst injection media*

    Separate into two 15 ml conical tubes, one with 10 ml of media and the other with 15 ml of media.

    Store on ice.

  4. Trypsinize the ES cell culture

    Add 1.0 ml trypsin.

    Incubate in tissue culture incubator for 10 minutes.

    Add 2 ml injection media*.

    Disperse cells thoroughly by pipetting with a P1000 pipet.

    Check cells under scope for good dispersion (we need single cells for injection).

  5. Place cells in 15-ml conical tube with 10 ml of the injection media prepared previously
  6. Place this ES cell suspension on ice
  7. Deliver the cell suspension (on ice) along with a separate tube containing the additional 13 ml of injection media (also on ice) to the GEMF core.

*Blastocyst Injection Media

  • 500 microliters 1M HEPES
  • 25 ml of blastocyst media

Blastocyst Media

  • DMEM + 10% Fetal Calf Serum
  • 2 mM Glutamine

Preparation of DNA for Electroporation into ES Cells

  1. Prepare at least 30 µg of the targeting vector plasmid. We recommend the Qiagen EndoFree Plasmid Maxi kit. Alternatively, one can use standard plasmid protocols followed by CsCl banding
  2. Linearize the targeting vector by digesting with a unique enzyme
  3. After digestion, check for complete linearization by running a small fraction on a mini-gel
  4. Precipitate DNA with two volumes of ethanol and 0.3 volume of 7.5 M ammonium acetate
  5. Wash pellet 2X with clean 70% ethanol
  6. Resuspend DNA in 0.1X TE* at concentration of 1.0 mg/ml. A total of 25 micrograms of linearized DNA is needed for electroporation.

*0.1X TE

Prepare 1X TE:

  • 10mM Tris-HCl, pH 8.0
  • 1mM EDTA, pH 8.0

Sterilize.
Dilute to 0.1X with sterile water.

Note:

  • It is very important to precipitate DNA with ammonium acetate rather than the commonly used sodium acetate
  • It is equally important to resuspend the DNA pellet in 0.1X TE according to the protocol above and not water or PBS
  • Not adhering to either of the above will greatly affect transformation efficiency

Preparation of DNA for Pronuclear Injection

  1. Prepare 30-50 µg of DNA. We suggest that you use a plasmid preparation protocol that results in endotoxin-free, clean plasmid DNA. The EndoFree Qiagen kit is suitable for this purpose.
  2. Digest enough transgene construct to release 20 µg of fragment to be injected. (The fragment to be injected has to be free of all vector sequences.)
  3. After digestion, check for complete digestion by running a dilute portion of the DNA on a gel. Compare to a lane of uncut plasmid and a lane where the digestion is overloaded (to check for additional unwanted products). Take a clear picture of this gel

Bring the entire digestion reaction to the facility for injection along with the picture of the gel, clearly indicating the fragment to be injected. The facility will further purify the fragment for injection.

Please contact the facility director for special preparation procedures for large DNAs (YACs, BACs, etc.)

Tail-DNA Extraction

  1. Place tail sample in 1.5-ml tube with 0.75 ml tail buffer*.
  2. Incubate at 55°C overnight with shaking.
  3. Extract digestion solution with equal volume of 1:1 phenol:choloroform then equal volume of chloroform.
  4. Precipitate DNA with equal volume of isopropanol.
  5. Spin down precipitate for 10 minutes.
  6. Invert tubes to discard supernatant.
  7. Dry pellet by placing tubes inverted over a paper towel at room temperature for an hour.
  8. Resuspend DNA pellet by adding 80 µl TE.

*Tail buffer

  • 100mM NaCl
  • 10mM Tris-HCl, pH 8.0
  • 25mM EDTA, pH 8.0
  • 0.5% SDS
  • 0.8 mg/ml Proteinase K

© 2014 The University of Texas MD Anderson Cancer Center