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International Journal Of Bilogical Sciences

.: Home > International Journal Of Bilogical Sciences > 2013 > Volume 9 Number 5 > Guijun Guan1,2, Yan Yan2, Tiansheng Chen2, Meisheng Yi3, Hong Ni2, Kiyoshi Naruse1, Yoshitaka Nagahama4,✉, Yunhan Hong2,✉

Nanos3 Gene Targeting in Medaka ES Cells

Guijun Guan1,2, Yan Yan2, Tiansheng Chen2, Meisheng Yi3, Hong Ni2, Kiyoshi Naruse1, Yoshitaka Nagahama4,✉, Yunhan Hong2,✉
1. Department of Bioresource, National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan; 2. Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543; 3. School of Marine Sciences, Sun Yat-Sen University, Guangzhou, 501275, China; 4. Institution for Collaborative Relations, Matsuyama 790-8577, Ehime University, Japan.
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.

Keywords :
ES, gene targeting, homologous recombination, nanos3, pluripotency.

Date Deposited : 11 Apr 2015 09:41

Last Modified : 11 Apr 2015 09:41

Official URL: http://www.ijbs.com/ms/archive

Volume 9, Number 5, - 2013 , ISSN 1449-2288

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