ES Cell Culture Services

We currently use J1 cells, a 129 line. We do have a C57 line as well, but we should discuss that if you want C57. If all goes well with the transfection, we expect to pick 192 clones and freeze these in 2 96 well plates at -80. For secondary Cre or Flp transfections, we pick 96 clones. About 3 weeks following the transfection, we give you 24 well plates of cells for you to lyse to make DNA and genotype. You should get enough DNA for at least one Southern and often two. PCR genotyping assays are the most convenient for this stage of genotyping, but these need to be confirmed by Southern eventually. Once you identify positive clones, we thaw up to 24 positives and karyotype up to 8 of them. We grow more cells for you to make more DNA for further genotyping at this point.

We strongly recommend that you give us the genotyping results within 4 months of the transfection. Longer storage at -80 decreases the success rate of thawing the clones.

When secondary Cre or Flp transfections are planned, we advise injection of the first round homologous recombinant lines before transfection as well, followed by crossing the resulting mouse lines to Cre or Flp expressing. This approach has proved very successful and these lines are used as backups in case the in vitro recombined lines fail to transmit germline. While we routinely do Cre transfections with excellent results, Flp is less efficient in vitro.

Repetitive DNA warning: Reduce (eliminate if possible) repetitive DNA sequences in the homologous arms of the targeting construct. Repetitive DNA greatly reduces the targeting efficiency.

Genotyping Information:

We request that people demonstrate that their genotyping assay for screening for homologous recombinants works on wild type ES cell DNA before having us do the transfection.

This is important because some probes surprise people with ugly smears indicating the presence of repetitive DNA in the fragment used for the probe. In some cases people have had to redesign their targeting vector to accommodate for a shifted probe fragment.

The requirements for proper genotyping assays are:

  • PCR and/or Southern confirmation of homologous recombination on both 5’ and 3’ ends of the targeting construct.
  • Southern probes that are external to the sequences used for the homologous arms of the targeting construct: i.e. probes must not be in the targeting construct.
  • An independent verifiable assay for any mutations remote from the drug selection cassette.
  • For PCR assays, one primer must be external to the homologous sequences in the targeting vector.
  • PCR assays must be demonstrated to work on extremely low copy number in the presence of genomic DNA.
  • PCR assays require the generation of a positive control plasmid that contains the external primer site as well as the change in the targeting vector. This control should be engineered to give a band of a size that differs from the ultimate homologous recombinant band to avoid cross- contamination confusion. An alternative approach to making a positive control plasmid involves designing primers that span the neomycin gene- ie. one external primer and one internal primer in the opposite homologous arm sequence. Demonstration that these primers work on wildtype DNA does not guarantee that they will work on the longer homologous recombinant DNA, however).

Please see “Genotyping Requirements” on the Policies page, point #3)

Please contact the Core to discuss genotyping further.

Before the transfection, we need to know:
What ES mouse strain line you want to target?
What are the positive and negative selections you need?
Will you want a secondary transfection with either Cre or Flp?
DNA prep for transfection:

  • Linearize 100 ug of DNA.
  • Prove that the linearization worked by running a small sample of the digest on a gel without ethidium bromide, next to a lane of uncut targeting vector.

Purify the digest by:

  • Phenol/Chloroform extraction,
  • Chloroform extraction (very important to get rid of the phenol),
  • Sodium acetate/ethanol precipitation.
  • Bring us the DNA in the sodium acetate/ethanol. We will resuspend and read OD...we transfect 50 ug.