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 We have formulated this hypothesis based on our published and unpublished data which demonstrate that simple cryopreservation methods such as freeze drying can preserve the genetic material without the requirement to maintain sperm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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01HD038205-03
Application #
6388174
Study Section
Special Emphasis Panel (ZHD1-DRG-A (12))
Program Officer
Rankin, Tracy L
Project Start
1999-09-01
Project End
2003-08-31
Budget Start
2001-09-01
Budget End
2003-08-31
Support Year
3
Fiscal Year
2001
Total Cost
$556,870
Indirect Cost
Name
University of Hawaii
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
121911077
City
Honolulu
State
HI
Country
United States
Zip Code
96822
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Ward, Monika A; Kaneko, Takehito; Kusakabe, Hirokazu et al. (2003) Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection. Biol Reprod 69:2100-8
Li, Ming-Wen; McGinnis, Lynda; Zhu, Liben et al. (2003) Intracytoplasmic sperm injection (ICSI) enables rescue of valuable mutant mouse strains. Comp Med 53:265-9
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Szczygiel, Monika A; Kusakabe, Hirokazu; Yanagimachi, Ryuzo et al. (2002) Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: a study with various mouse strains. Biol Reprod 67:287-92
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