PROJECT SUMMARY – GENE TARGETING AND TRANSGENIC FACILITY The Gene Targeting and Transgenic facility was founded over 28 years ago to assist AECC researchers in the generation of genetically modified mouse models of human disease. Initially, the Gene Targeting and Transgenic components operated as independent Shared Resources, however, following relocation to state-of- the-art laboratory space in the Price Center, the facilities were merged into a single Gene Targeting and Transgenic Shared Resource (GT&TF) In addition, a Gene Modification Service was implemented and integrated with the Facility to provide a complete service for the rapid and cost efficient generation of genetically modified mouse lines to all AECC investigators. The Transgenic component of the facility continues to generate, with high efficiency (~100% success rate), transgenic mouse strains through the introduction of DNA sequences, such as regular plasmid vectors or BAC clones, into the germ line by pronuclear injection or by lentivirus infection of fertilized oocytes. For each project, the facility supervisor consults with individual investigators and advises on transgene construct design, as well as making plasmids and sequence cassettes available to ensure suitability for expression in the mouse. The Gene Targeting component provided services for modification of the mouse genome through the use of CRISPR/Cas9 gene editing which was established during the current funding period. Although the vast majority of gene targeting projects are now conducted by CRISPR/Cas9, the facility continues to provide conventional gene targeting in embryonic stem (ES) cells and blastocyst injections upon request. Gene Targeting services include the generation of conventional knockout mouse lines, knock-in mouse lines and mouse lines with conditional alleles for the temporal and spatial ablation of genes. The facility has a high success rate (>95%) for obtaining gene targeted mouse lines. Finally, the services of the facility include the design and generation of simple and complex gene targeting vectors, electroporation and screening of embryonic stem cell lines as well as human and mouse cancer cell lines and the generation of chimeric mice by blastocyst injection. In addition, the facility offers mouse reproductive services for cryopreservation and re-derivation of transgenic and gene-targeted mouse lines as well as IVF services. Typically 25-30 investigators use this service each year with approximately 200 founder mice generated per year.