E1. OBJECTIVE A fundamental goal of this IDDRC is to understand the cellular and molecular mechanisms governing the normal development of the nervous system and identify the genetic basis for neuro-developmental disorders. One of the most powerful approaches toward this end is the creation of genetically defined mice that model the physiological effects of defined genetic alterations in the context of a living mammal. The specific objective of the Mouse Gene Manipulation Core is to provide all IDDRC investigators a centralized, affordable, quality-controlled service for the generation of genetically altered mouse lines, and to ensure that as insights into the genetic causes of human developmental disorders progress, mouse models for these can rapidly be made. The two categories of genetically altered mice widely used are those with targeted mutations, """"""""Knockout/Knock-in"""""""" mice, and those with random insertions of a foreign gene, """"""""Transgenic"""""""" mice. Examples of targeted mutations include full or partial gene deletions, replacements, splice site modifications, and amino acid changes. In addition, conditionally targeted mutations allow investigators to control the timing during development when a targeted mutation exerts its effect, and also allow the analysis of mutations directed to specific cell types or tissues. Transgenes can encode over- or mis-expressed wild-type or mutant proteins or inhibitory RNA molecules (RNAi). The mutant proteins or RNAs can either be inappropriately active or act as dominant negative suppressors of normal endogenous protein activity. Gene targeting exploits the pluripotency of mouse embryonic ES cells which, when injected into host mouse embryos, are capable of generating germ cells (sperm and eggs) that can pass their genetic content on to subsequent generations. ES cells are manipulated in culture to alter endogenous ES cell genes with specific mutations. These """"""""targeted"""""""" ES cells are then injected into mouse embryos that are grown to fully mature mice. ES cells contribute to the development of the germ cells, and when the mating of these mice result in the passage of the targeted mutation to the next generation, a novel mouse line is established. For transgenic mice, transgenic DNA sequences are microinjected directly into the pronuclei of single-cell embryos. In a subset of the injected emtiryos, the injected DNA will stably and randomly insert into the mouse genome and can be transmitted across generations. Of the technologies required for the generation of genetically altered mice, the culturing and genetic modification of embryonic stem (ES) cells, micro-manipulation of preimplantation mouse embryos, and embryo transfer mouse survival surgeries are best fulfilled by a core facility. ES cell culture requires quality-controlled reagents and highly trained staff. Microinjection and mouse surgery require specialized skills, equipment, and a dedicated mouse colony. The costs of developing and maintaining the ability to perform these procedures are prohibitive to most investigators, especially those that need to generate new mouse lines infrequently. Thus, the consolidation of these technologies within a core facility serving multiple investigators allows individual investigators access to these specialized techniques that might otherwise not be available to them. To this end the IDDRC Mouse Gene Manipulation (MGM) Core exists to ensure that all IDDRC investigators have the opportunity to use genetically altered mice to advance their research into developmental neurobiology and the neural diseases allied to the mission of the Center. This Core is a centralized, state-of-the-art and cost-effective service that performs specialized technologies critical for the generation of genetically altered mice. The Core maintains the necessary specialized equipment, reagents, and mouse colonies. The skills of the Core staff cover all the procedures involved in ES cell culture, mouse embryo microinjection and surgery, as well as in maintenance of the requisite animal colonies. The Core also is a centralized resource for reagents, protocols and consultation for the design and construction of DNA vectors and genotyping assays, and the maintenance and analysis of novel mouse lines. The Core has overseen many projects involving a wide variety of experimental approaches, and is thus a repository of a large collective experience readily available to all IDDRC investigators to aid them in the design and utilization of the most suitable mouse models for their projects. Finally, the Core is dedicated to following technological developments in the field and is well suited to introduce new techniques that will advance the research goals of IDDRC investigators.
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