Services and Pricing

Gene Targeting

A gene targeting experiment is a multistep undertaking that requires coordination and cooperation between the Principal Investigator (PI) and this resource.

1. First, the PI must design, assemble and correctly annotate a gene targeting vector that will achieve the desired experimental objectives. This should be done in a manner compliant with our guidelines for the Design and Assembly of Targeting Vectors. An essential part of the design phase is to identify and test restriction sites and Southern blot probes that will be used to identify those mouse embryonic stem cells (mESCs) that have undergone homologous recombination. When the PI is certain that the vector has been correctly assembled and documented, and that the screening strategy is certain to work, s/he should then submit a Gene Targeting Service Form containing all requested information. In addition, the PI should download, review and sign the Operational Policies Form.
2. The completed form will be then be reviewed by both the Managing and Scientific Directors of the resource to ensure that the strategy is solid, and the vector has been correctly documented. If the experiment is judged to be adequately designed and documented it will be scheduled and started as soon as possible.
3. After electroporation of the targeting vector into a mESC cell line at least 200 resistant clones will be picked and grown in triplicate. One set of colonies is placed in short term storage at -70°C, and DNA is isolated from the other two plates. DNA from one of these two plates is used for Southern blot analysis. The other serves as a back-up source of DNA. Southern blots may either be prepared by the personnel of this resource, or by the investigator. The primary screen of the mESC clone DNAs must be completed within 4 months to assure that cells remain viable.
4. After screening results are reviewed, and both the PI and Managing Director agree on which clones are potential positives, up to eight clones are thawed, expanded and frozen in liquid nitrogen. Additional DNA is prepared from these clones, and given to the PI who is then responsible for confirming that the gene targeting event is correct by performing additional Southern blot hybridizations and DNA PCR analysis. Because cells are frozen in 96 well dishes, they cannot be thawed and refrozen. For this reason all non-targeted clones are discarded.
5. Upon confirmation of correct homologous recombination, at least one mESC clone is chosen for microinjection into blastocysts derived from timed matings of C57Bl/6 mice. Chimeric males resulting from the blastocyst microinjection experiments are provided to the investigator following serology testing by the Division of Animal Care. This usually occurs at about 7 weeks of age. The PI is then responsible for mating these animals and reporting back to the resource the number of pups generated from each chimera and germ line transmission results.

Given the substantial, and not always successful, effort involved in a gene targeting experiment, half of the cost of the experiment is billed in advance to cover the cost of supplies and personnel effort regardless of the outcome. The remainder of the service fee is charged when germline transmission is achieved. In cases where very few or no targeted cells are identified, and the gene targeting experiment needs to be repeated, additional charges will be assessed.