? The rapid advances in molecular genetics and reproductive technology during the past decade have led to the production of large numbers of artificially produced animal genotypes. The synergy of these two fields has been especially influential in the mouse, since this species is now the primary animal model for many human diseases. It is important, therefore, to preserve the large numbers of mouse genotypes for use by future investigators as economically as possible. The traditional way of storing mouse genotypes is by the maintaining of breeding colonies. However, the cost of maintaining many mouse-breeding colonies has become prohibitively expensive. 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 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 of sperm offers a simple and inexpensive approach that can be mastered quickly, and accomplished successfully with minimal training. Furthermore, 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.
In Specific Aim 1, we will optimize evaporative drying protocols for long-term, ambient temperature storage of mouse sperm from three inbred strains and from three genetically altered mutant lines.
In Specific Aim 2, we will develop an automated intracytoplasmic sperm injection (ICSI) procedure that is inexpensive and satisfies the safety requirements that prohibit the use of highly toxic and regulated metallic mercury.
In Specific Aim 3, we will carry out phenotypic analysis of embryos and offspring generated by evaporatively dried sperm after experiencing a wide variety of conditions that simulate storage and transport. When the proposed studies have been completed, we expect the overall outcome to be the establishment of a simple and reliable method for mouse sperm evaporative drying, and verification that ICSI of dried sperm can be used successfully to propagate mouse strains. ? ? ? ? ?
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