Archiving biomedical mouse models by ovary cryopreservation

The use of genetically modified (GM) mice for biomedical research has tremendously increased since 1980’s. Housing the rapidly expanding number of mouse lines has become a problem and specific lines were even accidentally lost due to the lack of space or loss of fertility during breeding for maintenance. Cryopreservation is a strongly recommended approach to solve these problems, also from an ethical point of view. There are also technical risks by small-scale breeding, including genetic drift, microbiological or genetic contamination, disease, or large-scale disasters. The investigations presented in this thesis focus on cryopreservation of mouse ovaries to maintain the expanding populations of GM mice, and the target strains (C57BL/6, FVB, BALB/c) utilized are those commonly used as the genetic background. The major aim of the research is to develop an efficient, economic and ethically sound cryopreservation and validation protocol for archiving the mutant mouse lines. In chapter 2, a comparison is made of the currently used technologies for cryopreservation of germplasm in mice and the subsequent retrieval of these mutant lines. Detailed managerial information concerning animal use, and time and cost related to these technologies is evaluated. In chapter 3, a cryopreservation protocol for mouse ovaries using high concentrations of cryoprotectants and instrumented ultra-rapid freezing for vitrification is developed and validated by ovary transplantation and subsequent breeding. The robustness of the developed freezing protocol is investigated from various genetically modified lines with different genetic backgrounds (chapter 4). The over-time functionality of the transplanted ovaries is investigated with regard to efficiency in delivering mutant offspring. In chapter 5, the in vivo development of cryopreserved ovarian tissue is investigated by in vivo imaging after allotransplantation. The optimal donor age is also evaluated. The use of a spontaneous albino mutant of the (black) C57BL/6 as a recipient is also evaluated as a rapid method for identifying the parentage of delivered pup. In chapter 6, the application of transplantation of fresh grafts is evaluated in propagating a subfertile mutant mouse line and the reduced reproduction efficiency related to a particular genetic modification is investigated. Specific attention is focused on the origin of the abnormality. Finally, an overview of the results presented in these studies and their possible implications for future practice and researches are discussed in chapter 7. “There is really no limit to the number of mutant mouse strains that could be generated and expanded to infinity” is practically true in the filed of mouse genetics. Thus, how to manage the mouse colonies in a responsible way is an urgent issue and is an obligated responsibility of researchers. In this thesis, we present the practical information about cryopreservation of germplasm for the management of mutant mouse colonies. The successful application on cryopreservation of juvenile ovaries provides an instant solution to animal facilities that are facing space problems of housing mouse colonies and may also provide useful information to cryopreservation of human ovarian tissue, serving as a model for autotransplantation and studying graft/host interactions in a variety of models for human diseases.