1) CRISPR/Cas9 Gene Targeting in Fertilized Eggs. Cas9 protein, guide RNA and targeting vector are injected into fertilized eggs to generate knockout or targeted mutations. This new technology generates mutant mice faster, cheaper and more reliably than conventional targeting.
2) Transgenic Mice. The facility offers zygote pronuclear injection service for the generation of transgenic mice. This includes a wide variety of transgenes, including classic promoter plus cDNA constructs, doxycyline-regulated transgenes and large genomic fragments (BAC transgenics). Our core is one of the few transgenic cores with an extensive success record with BAC transgenics, including BAC transgenics on the more difficult C57BL/6J inbred background.
3) Gene Targeting in Embryonic Stem Cells. Gene targeting is performed in either 129 or C57Bl/6N embryonic stem cells. This includes knockout (null), knockin, point and conditional (floxed) mutants. Our gene targeting service began in 2001, and thus is one of the longest running and most experienced in the country.
4) Chimeras. The core offers blastocyst injection of embryonic stem cells to generate chimeric mice for germ line transmission of targeted mutations. The core injects cell lines that were targeted in the core, as well as cells obtained from public repositories. We also make aggregation chimeras and ES/tetraploid chimeras.
6) In Vitro Fertilization and Frozen Embryo Transfer. The latest technologies for in vitro fertilization are used. Reanimation of cryopreserved embryos from public repositories or other labs can be done by frozen embryo transfer.
7) Generation of New Embryonic Stem Cell Lines. The facility generates new embryonic stem cell lines from investigators' mutant mice.
8) Rederivation of Mice. The facility performs embryo transfer into pseudopregnant females to rederive strains of mice to specific-pathogen-free status. The core works closely with the Associate Director of the Animal Resource Center in the pathogen control program.
9) Ovariectomy, Ovary Transplantation, and Vasectomy. The facility performs some minor surgeries.
The Case Transgenic and Targeting Facility generates transgenic and knockout mice. The facility also performs rederivations, in vitro fertilization, cryopreservation, generates chimeric mice and makes mutant mice with CRISPR/Cas nucleases. It provides consultation on scientific, technical and practical matters of mouse genetics. It is the only transgenic core in the Cleveland area, and serves investigators at CWRU, UHCMC, CCF, the VA Hospital, Metro-Health Hospital and Kent State University. The Transgenic Facility is competitive with transgenic cores at other academic institutions in success rates, turnaround and pricing. As a local service, it has advantages for local researchers in terms of direct access to consultation and training, in better pricing of services and in control of mouse pathogens.
The transgenic core places a high priority on consultation, education and training and provides them free of charge for all investigators. Consultation, education and training are carried out by the core director, the core staff, and by the core web site.
Consultation: Scientific and strategic consultations with the core director begin with discussion of the scientific goals and the best means of meeting those goals, whether through the creation of new transgenic or mutant mice, or through the use of existing strains from public resources. Investigators and their staff vary greatly in their level of expertise and experience with mouse genetics: clients with little or no experience are strongly encouraged to meet with the core director for scientific consultation.
Education and Training: The core director and staff train investigators in mouse genetics and biology and in the practical aspects of breeding and genotyping mice. The practical aspects are taught to investigators and their staff through direct interaction and through the core's web site. The practical information ranges from how to sex mice to the appropriate controls for tissue-specific knockouts.
A large amount of relevant and up-to-date original information is provided on the facility web site. The core web site was created and is maintained by the core director, Dr Conlon. The site hosts a wealth of original information about mouse biology, genetics and breeding, protocols, conceptual information about which genetic technologies are best suited for experimental goals, the design of transgene and gene targeting vectors, and the timeline, costs, investigator effort and expected results for each technology.
Customer Service: When orders are placed, a core staff member contacts the client by to guide them and request any necessary materials (maps, DNAs, validation of genotyping assays). Clients are sent a link to a tracking page on the web which keeps them updated on the progress of the job through automatic updates from the core database.