RRC ID 30924
Author Guan G, Yan Y, Chen T, Yi M, Ni H, Naruse K, Nagahama Y, Hong Y.
Title Nanos3 gene targeting in medaka ES cells.
Journal Int. J. Biol. Sci.
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.
Volume 9(5)
Pages 444-54
Published 2013
DOI 10.7150/ijbs.6507
PII ijbsv09p0444
PMID 23678294
PMC PMC3654493
MeSH Animals Blotting, Southern Embryonic Stem Cells / metabolism* Gene Targeting / methods* Genetic Engineering / methods* Genetic Vectors / genetics Genotype Mice Oryzias / genetics* Plasmids / genetics Pluripotent Stem Cells / metabolism* RNA-Binding Proteins / genetics* Reverse Transcriptase Polymerase Chain Reaction Sequence Analysis, DNA Species Specificity
IF 4.057
Times Cited 4
WOS Category BIOLOGY BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Medaka database