Gene Targeting Protocols

Submitting DNA for Electroporation Protocol

  1. Clients may discuss the design of the gene targeting experiments with the Germline Modification Core staff.
  2. Clients are responsible for isolating an isogenic, genomic DNA clone from a 129/SvJ library or other suitable source.
  3. This clone will need to be restriction mapped, sequenced in critical regions and engineered to delete critical sequences and contain selection markers.
  4. It is desirable to have at least 7 kb of homology to the endogenous gene for efficient homologous recombination to occur. To facilitate PCR analysis of ES cell clones and mice, one arm of homology should be approximately 1.5 - 2.0 kb in length.
  5. The client must provide evidence of a successful strategy to identify positive homologous recombination events by both Southern blot analysis and PCR. This evidence shall consist of construct maps as well as PCR and Southern blot results showing the expected endogenous bands.
  6. Probe sequences used to identify homologous recombination events must be located outside the genomic sequences used to create the targeting vector. These probes, when hybridized to a Southern blot of restriction enzyme digested,129/SvJ genomic DNA, should identify a single copy of the targeted gene (the endogenous gene).
  7. Once the construct is complete and genotyping strategies approved, the client will submit 100 µg of linearized, purified vector DNA at a concentration of 1 µg/µl in sterile, embryo tested water (Ultrapure Water, Specialty Media Cat.# TMS-006-A) 
  8. The targeting vector DNA should be shipped to the Germline Modification Core by overnight shipping on dry ice to:

    Audra Guikema
    Van Andel Research Institute
    333 Bostwick, NE
    Grand Rapids, MI 49503

Submitting ES cells for Microinjection Protocol

  1. Clients may wish to provide gene targeted ES cells to the Germline Modification Core for microinjection into blastocysts and production of chimeras.
  2. These cells must be pathogen tested prior to shipping to the facility.  Please visit the University of Missouri's Web site external link for information on their pathogen testing service.
  3. Clear instructions as to the growth and selection history of the cells must be outlined prior to shipping the cells. Prior consultation with the facility is required for these protocols.

DNA Isolation from Mouse Tissue Protocol

  1. Excise ~0.5 cm of the mouse tail tip and place in a 1.5 ml eppendorf tube.
  2. Add 400 µl of tissue digestion buffer (10 mM Tris, pH 7.5, 10 mM EDTA, 100 mM NaCl, 0.5% SDS) containing 50 µg/ml Proteinase K (Roche Molecular)
  3. Incubate overnight at 55°C with gentle shaking.
  4. Allow the tubes to cool to room temperature and flash spin to remove the solutions from the tube lid.
  5. Add 400 µl of phenol/chloroform solution (1:1).
  6. Close the tubes and mix vigorously by shaking or inverting the tubes (do not vortex).
  7. Spin in a microfuge at maximum speed for 5 minutes.
  8. Transfer the top aqueous phase to a new 1.5 ml eppendorf tube. Use a large orifice pipetman tip to transfer.
  9. Precipitate the DNA by adding 2 volumes of absolute ethanol and shake vigorously until the precipitate is visible.
  10. Spin in a microfuge at maximum speed for 4 minutes. Decant the supernatant.
  11. Wash the pellet with 200 µl of 70% ethanol.
  12. Spin in a microfuge at maximum speed for 1 minute. Decant all of the supernatant.
  13. Air dry the tubes.
  14. Resuspend the DNA pellet in 200 µl TE buffer (10 mM Tris, 1 mM EDTA). Resuspension of the DNA can be aided by incubating the tubes at 37°C for several hours.

Breeding Protocols

  1. Chimeric mice must be tested for germline transmission of the mutant allele.
  2. Since the ES cells are male and derived from mice with an agouti coat color, most chimeras are male with a substantial amount of coat color contribution from the agouti ES cells.
  3. Male chimeras that are >75% agouti should be bred when they are 7-8 weeks of age.
  4. Male chimeras should be individually mated to 2 C57BL/6 females, 7-8 weeks of age.
  5. Females should be placed in the male's cage either alone or in pairs.
  6. The female mice should be checked each day for the presence of a copulation plug indicating that mating has occurred.
  7. Females should be removed from the mating cage to gestate in individual cages.
  8. Genotype analysis will be performed on DNA from tail biopsies of the offspring.
  9. Mice heterozygous for the gene targeted allele are intercrossed at 7-8 weeks of age and the resulting litters genotyped.
  10. Mice homozygous for the gene targeted allele should be identified in the normal Mendelian ratio expected for a recessive genotype.
  11. If homozygotes are not identified after several litters, it is likely that the mutants are embryonic lethal or die at birth. In this case, timed matings should be set up and embryos isolated at various embryonic stages for phenotype analysis. Yolk sac DNA can be isolated from each embryo for genotype analysis.