title: Production of loach (Misgurnus anguillicaudatus) germ-line chimera using transplantation of primordial germ cells isolated from cryopreserved blastomeres.
abstract: An efficient procedure for the cryopreservation of fish blastomeres followed by restoration through germ-line chimera formation was established. Blastomeres of the loach (Misgurnus anguillicaudatus) were cryopreserved in 250-µL straws in Eagle's minimum essential medium with various concentrations of dimethyl-sulfoxide (0, 5, 10, 15, and 20%), and the best concentration was combined with glycerol (1, 2, and 4%) and external cryoprotectants (1 or 2% sucrose; 2, 5, or 10% fetal bovine serum; 1 or 2% BSA). Postthaw viability of the blastomeres was used to optimize cryopreservation conditions. Donor blastomeres were injected with zebrafish green fluorescence protein-nos1 3' untranslated region mRNA and biotin dextran before cryopreservation in the optimal freeze medium. Host embryos were injected with zebrafish DsRed-nos1 3' untranslated region mRNA and reared to the blastula stage. Donor blastomeres were thawed at 25 °C for 10 s and transplanted to the host embryos either immediately or after incubation for 16 h at 20 °C. Donor and host primordial germ cell migration was visualized with fluorescent imaging during the early stages of embryogenesis, and also by histology in 4-d-old embryos. Transplantation of blastomeres immediately after thawing gave decreased hatching rates (approximately 3%) and generated a smaller percentage of germ-line chimeras (approximately 1.1%). In contrast, incubation of a cryopreserved sample for 16 h followed by transplantation of the green fluorescence protein-positive blastomeres improved the hatching rate to 90%, and successfully produced presumable germ-line chimeras at a rate of 16.5%. The improved survival rates and germ-line chimerism may be an effective method for gene banking and subsequent reconstitution of endangered fish genotypes.
authors.affiliation: Laboratory of Aquaculture Genetics and Genomics, Division of Marine Life Sciences, Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1, Hakodate, Hokkaido, 041-8611, Japan. george@fish.hokudai.ac.jp
authors.forename: G S
authors.initials: GS
authors.lastname: Yasui
authors.forename: T
authors.initials: T
authors.lastname: Fujimoto
authors.forename: S
authors.initials: S
authors.lastname: Sakao
authors.forename: E
authors.initials: E
authors.lastname: Yamaha
authors.forename: K
authors.initials: K
authors.lastname: Arai
mesh_terms: D000818:Animals; D001757:Blastomeres / Q000166:cytology* / Q000502:physiology*; D018929:Cell Culture Techniques; D002678:Chimera; D015925:Cryopreservation / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D003531:Cypriniformes / Q000502:physiology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005854:Germ Cells / Q000166:cytology* / Q000502:physiology*; D005990:Glycerol / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013395:Sucrose / Q000494:pharmacology
chemical_list: D003451:Cryoprotective Agents; D003470:Culture Media; D012710:Serum Albumin, Bovine; D013395:Sucrose; D005990:Glycerol; D004121:Dimethyl Sulfoxide