We propose to purchase a Zeiss microinjection station for use in the University of Utah Transgenic and Gene Targeting Mouse Core. This core facility is heavily used by NIH- funded investigators, and has contributed to many of their published manuscripts. The capabilities of the instrument and its associated hardware include injection of mouse embryonic stem cells into blastocysts, injection of DNA into the pronuclei of fertilized eggs, 8-cell stage injections of embryonic stem (ES) cells, assisted in vitro fertilization (AIVF), and intracytoplasmic sperm injection (ICSI). Many of the procedures used to generate transgenic mice were developed at the University of Utah, and we have one of the oldest core facilities. The downside is that our microinjection instrument is now 20 years old, and lacks many features essential for highly efficient mouse generation. Production of gene-targeted mice and the generation of transgenic mice via pronuclear injection are essential parts of mammalian research. Because of the technical expertise required, the generation of gene targeted and transgenic mice is usually performed in a Core facility such as ours. Having updated, state-of-the-art equipment that is used every day in the Core immensely increases the success and efficiency of projects for researchers. Most gene-targeted mice are generated by microinjecting targeted embryonic stem cells into blastocyst stage embryos. This is done using a powerful microscope and micromanipulators to select and inject individual ES cells into each blastocyst. Injected blastocysts are surgically implanted into pseudopregnant females which carry the embryos to term. Pups born are chimeras which can pass on the targeted gene to its offspring. 100% germline gene-targeted mice can also result from injecting ES cells into 8-cell stage embryos. Excellent optics and micromanipulators are essential to these procedures. Transgenic mice are produced by injecting manipulated DNA into the pronucleus of fertilized eggs. DNA integrates into the genome randomly and is useful if using a tissue-specific promoter to drive or overexpress a gene of interest or reporter. AIVF (assisted in vitro fertilization) is performed by boring a hole in the zona pellucida of an unfertilized egg using a laser, which makes it easier for sperm to fertilize the egg, immensely improving results. ICSI (intracytoplasmic sperm injection) is used to directly inject immobile sperm into an egg. This procedure is used if normal IVF or AIVF is unsuccessful. It is imperative that the injector have excellent optics in this procedure.
