To define the functions of genes and cell types in vivo, there is no substitute for the power of modifying the mouse germline to generate gene knockouts (KO) and mutants or to express new genes transgenically. For optimal interpretation, genes need to inactivated conditionally or only in certain tissues, or expressed as transgenes in a tissue specific manner in the native genomic context. Using these techniques, investigators worldwide and particularly at Yale, have already been making numerous KO, knockin, transgenic, etc. mouse strains and breeding the modified alleles onto useful genetic backgrounds. These strains, many of which are published, are invaluable resources for research in Rheumatologic diseases, but the maintenance of them is time consuming and expensive, and one cannot envision the endless collection of more and more strains of actively breeding mice. Moreover, infection and breeding problems threaten the existence of many strains, and make the transfer among investigators cumbersome and costly. At the same time, to date the vast majority of engineered mutations have been made by a relatively small number of investigators;these technologies need to be more widely available so that labs that focus on particular genes or processes can have direct access to modifying them, instead of relying on labs that specialize in making KO mice. An important barrier to the technology is that the process is expensive, difficult to master, fraught with potential pitfalls, and time-consuming. In addition, it is limited to targeting ES cells of 129/Sv or (with more difficulty) B6;autoimmune prone strains cannot be directly modified. To address these issues, Core B will undertake 3 Aims. First, to make gene targeting accessible to YRDRCC members, the Core will provide introduce the novel TALEN endonuclease approach to genetic modification of the mouse germline. It allows the targeting of any gene directly in fertilized eggs, without the need for complex constructs (a simple PCR fragment is used), without the need for unwanted selectable markers, and without any ES cell work. TALEN technology can be applied to any strain, allowing us to directly modify autoimmune-prone animals. Most importantly, it is much faster, simpler, and cheaper, which will allow many more investigators to use the approach. Second, the Core will provide cryopreservation capabilities for the cost-effective storage of genetically modified mice. In our first funding cycle we froze sperm from over 380 strains. However, we did not preserve embryos from strains with multiple modified traits, leaving many very valuable lines unpreserved. We now plan to freeze these strains as embryos. Additionally, the core will continue cryopreservation of sperm, which is still required by many investigators. This will produce substantial decreases in costs associated with preservation of mouse strains. Finally, the Core will promulgate a web database of our 380 (and growing strains), facilitate their distribution to others, and in addition begin to freeze sperm of key strains from non- Yale investigators. This Core is thus both innovative and of high impact within and outside of Yale.
Genetically modified animals are the mainstay of modern immunologic research, and are key to identifying mechanisms of disease and how to design drugs to treat them. This Core will enable Yale and non-Yale researchers to more easily make new types of mice, to test new ideas of mechanism and therapy, while better preserving and distributing a vast library of existing strains.
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