Services and Pricing

Gene Targeting

This procedure enables specific genetic modifications such as conditional, knock-in or null alleles to be introduced into the mouse genome. 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 the 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.
2. The completed form will 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. Upon approval of the project, linear DNA provided by the investigator is electroporated into mESCs. Procedures for the targeting vector DNA prep are available on our website. The cells are then cultured under appropriate selection (hygromycin-B, G418, puromycin, and/or gancyclovir). Surviving colonies are 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, 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-desired clones from that 96-well plate 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.

Experimental Flow:

1. PI submits DNA and appropriate service form to TMESCSR for electroporation
2. Service forms are reviewed and approved by the Co-Directors of the resource
3. DNA is prepared by the PI.s laboratory and submitted to the resource. Note . because this is linear DNA, it should be frozen at -20oC and delivered to the TMESCSR on dry ice.
4. Electroporation is scheduled, cells are prepared for the experiment
5. Electroporation performed, cells are under selection for ~7 days before picking of individual colonies
6. Colonies are picked, grown in triplicate
7. Southern analysis of clone DNA
8. PI identifies correctly targeted clones
9. Targeted clones are grown and expanded and frozen back in LN2
10. Additonal clone DNA is provided to the investigator for rescreening
11. PI confirms correctly targeted clones and requests ES cell microinjections