The rapid advances in molecular genetics and reproductive technology during the past decade have led to the production of a large number of artificially produced animal genotypes. The synergy of these two fields has been especially influential in the mouse, since it is now the primary animal model for many human diseases. It is important, therefore, to reliably preserve the large numbers of mouse genotypes for use by future investigators as safely, simply, and economically as possible. Traditionally, mouse genotypes are stored by maintaining active breeding colonies of mutant mice. However, the cost of live colony maintenance has become prohibitively expensive. Alternatively, the banking of spermatozoa would be an efficient and cost effective approach for the storage of transgenic and mutant stocks. Several methods are available today for cryopreservation and maintenance of mouse sperm, but these require sophisticated freezing equipment and methodologies. In all cases, ultra low temperature storage in liquid nitrogen is required. It is also difficult and expensive to ship frozen sperm. Evaporative drying offers a simpler, faster, and more inexpensive approach that can be mastered quickly and accomplished successfully with minimal training. Furthermore, cellular stability is achieved quickly at ambient temperature without prolonged exposure to deleterious conditions during drying. Organisms that naturally enter a dried state during dormancy do so by evaporative drying, which suggests evaporative drying may be innocuous to cells under proper conditions. If this technique could be applied to mouse sperm, then the steadily increasing number of mutant mouse lines could be reliably preserved and stored at minimal cost and for maximum benefit.
In Specific Aim 1, we will develop a simple, reproducible, and low-cost rapid drying technique for long-term, room temperature storage of mouse sperm from three inbred strains and from three genetically altered mutant lines.
In Specific Aim 2, we will achieve high-throughput intracytoplasmic sperm injection (ICSI) production of mice from evaporatively-dried sperm in the absence of mercury by development of a completely automated and novel microinjection technology.
In Specific Aim 3, we will analyze the viability, health, and genetic and phenotypic fidelity of embryos and live born offspring generated from evaporatively dried sperm stored at ambient temperatures. ? ? ?
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