title: Nanos3 gene targeting in medaka ES cells.
abstract: Gene targeting (GT) by homologous recombination offers the best precision for genome editing in mice. nanos3 is a highly conserved gene and encodes a zinc-finger RNA binding protein essential for germ stem cell maintenance in Drosophila, zebrafish and mouse. Here we report nanos3 GT in embryonic stem (ES) cells of the fish medaka as a lower vertebrate model organism. A vector was designed for GT via homologous recombination on the basis of positive-negative selection (PNS). The ES cell line MES1 after gene transfer and PNS produced 56 colonies that were expanded into ES cell sublines. Nine sublines were GT-positive by PCR genotyping, 4 of which were homologous recombinants as revealed by Southern blot. We show that one of the 4, A15, contains a precisely targeted nanos3 allele without any random events, demonstrating the GT feasibility in medaka ES cells. Importantly, A15 retained all features of undifferentiated ES cells, including stable self-renewal, an undifferentiated phenotype, pluripotency gene expression and differentiation during chimeric embryogenesis. These results provide first evidence that the GT procedure and genuine GT on a chromosomal locus such as nanos3 do not compromise pluripotency in ES cells of a lower vertebrate.
authors.affiliation: Department of Bioresource, National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan.
authors.forename: Guijun
authors.initials: G
authors.lastname: Guan
authors.forename: Yan
authors.initials: Y
authors.lastname: Yan
authors.forename: Tiansheng
authors.initials: T
authors.lastname: Chen
authors.forename: Meisheng
authors.initials: M
authors.lastname: Yi
authors.forename: Hong
authors.initials: H
authors.lastname: Ni
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Naruse
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Nagahama
authors.forename: Yunhan
authors.initials: Y
authors.lastname: Hong
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D005838:Genotype; D051379:Mice; D009990:Oryzias / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013045:Species Specificity
chemical_list: C477342:Nanos3 protein, mouse; D016601:RNA-Binding Proteins
keywords: ES; gene targeting; homologous recombination; nanos3; pluripotency.