Molecular biologists have developed the technology to make precise additions, deletions or alterations to the genetic material of experimental animals. The mouse is the species which has been most widely studied, and genetically altered mouse models are being used for investigation of a wide variety of normal biological processes and diseases. Each of these models is a unique resource which is worthy of preservation, but the number of strains that is being generated is now surpassing the capability of the scientific community to maintain them. The contemporary approaches of creating breeding colonies and banks of frozen embryos must be augmented by methods that are relatively inexpensive and less labor-intensive. It is now acknowledged that cryopreservation of mouse sperm is the preferred approach for storing genetically altered mouse strains for subsequent reconstitution. It is also recognized that despite many years of research effort, little progress has been made in developing cryobiology techniques capable of preserving large numbers of living, fertilization-competent mouse sperm, and this significant problem is likely to be exacerbated by significant variability in the cryoprotection requirements of sperm from genetically altered strains. The objective of this particular application is to optimize protocols for cryopreservation and ICSI of sperm from genetically altered strains of mice including transgenic strains, knockout strains and strains that have been altered by ENU mutagenesis, to confirm the passage of the altered genotype and to assess the phenotype of the reconstuituted strains in terms of their fertility and incidence of pathology. The central hypothesis for the proposed research is that sperm from genetically altered strains of mice can be cryopreserved for long periods of time without alteration of their genetic material. We have formulated this hypothesis based on our published and unpublished data which demonstrate that simple cryopreservation methods such as freeze drying can preserve the genetic material without the requirement to maintain sperm viability. The rationale for focusing this application on the combined approaches of simple sperm cryopreservation and ICSI is that, such methods are likely to be applicable to sperm from a wide variety of mouse strains with little or no modification, and that they can be easily learned and applied in other centers.
