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Production of loach (Misgurnus anguillicaudatus) germ-line chimera using transplantation of primordial germ cells isolated from cryopreserved blastomeres

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Title: Production of loach (Misgurnus anguillicaudatus) germ-line chimera using transplantation of primordial germ cells isolated from cryopreserved blastomeres
Authors: Yasui, G.S. Browse this author
Fujimoto, T. Browse this author
Sakao, S. Browse this author
Yamaha, E. Browse this author →KAKEN DB
Arai, K. Browse this author →KAKEN DB
Keywords: Aquaculture
Germ cell
Issue Date: 1-Aug-2011
Publisher: American Society of Animal Science
Journal Title: Journal of animal science
Volume: 89
Issue: 8
Start Page: 2380
End Page: 2388
Publisher DOI: 10.2527/jas.2010-3633
PMID: 21398566
Abstract: An efficient procedure for 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 (MEM) with various concentrations of dimethyl-sulfoxide (DMSO; 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% bovine serum albumin). Post-thaw viability of the blastomeres was used to optimize cryopreservation conditions. Donor blastomeres were injected with zebrafish GFP-nos1 3’UTR mRNA and biotin dextran prior to cryopreservation in the optimal freeze medium. Host embryos were injected with zebrafish DsRed-nos1 3’UTR 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 lower hatching rates (~3%) and generated a low percentage of germ-line chimeras (~1.1%). In contrast, incubation of cryopreserved sample for 16 h followed by transplantation of the GFP-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.
Type: article (author version)
Appears in Collections:水産科学院・水産科学研究院 (Graduate School of Fisheries Sciences / Faculty of Fisheries Sciences) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 荒井 克俊

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