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Cryopreservation of green fluorescent protein (GFP)-labeled primordial germ cells with GFP fused to the 3' UTR of the nanos gene by vitrification of Japanese eel (Anguilla japonica) somite-stage embryos.
MedLine Citation:
PMID:  22829617     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Primordial germ cells (PGC) are the only cell type in developing embryos with the potential to transmit genetic information to the next generation. In this study, PGC of Japanese eel (Anguilla japonica) were visualized by injection of mRNA synthesized from a construct carrying the green fluorescent protein (GFP) gene fused to the 3' untranslated region of the Japanese eel nanos gene. We investigated the feasibility of cryopreserving Japanese eel PGC by vitrification of dechorionated whole somite-stage embryos. The GFP-labeled PGC were rapidly cooled using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (methanol, dimethyl sulfoxide, and glycerol for 10 min and ethylene glycol for 10, 20, and 30 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose for 1, 5, and 10 min. Ethylene glycerol is an effective cryoprotectant for embryonic cells and shows no evidence of ice formation after thawing. Vitrified/thawed PGC were transplanted into blastula-stage embryos from zebrafish (Danio rerio). The GFP-labeled PGC migrated toward the host gonadal ridge, suggesting maintenance of their normal migration motility. These techniques may assist in achieving inter- and intra-species germ-line chimers using donor Japanese eel PGC.
Authors:
Y Kawakami; M Ishihara; T Saito; T Fujimoto; S Adachi; K Arai; E Yamaha
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-7-24
Journal Detail:
Title:  Journal of animal science     Volume:  -     ISSN:  1525-3163     ISO Abbreviation:  -     Publication Date:  2012 Jul 
Date Detail:
Created Date:  2012-7-25     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8003002     Medline TA:  J Anim Sci     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Nanae Fresh Water Laboratory, Field Science Center of Northern Biosphere, Hokkaido University, Nanae 041-1105, Japan.
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