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Nanos3 Gene Targeting in Medaka ES Cells
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 ste...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Ivyspring International Publisher
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654493/ https://www.ncbi.nlm.nih.gov/pubmed/23678294 http://dx.doi.org/10.7150/ijbs.6507 |
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author | Guan, Guijun Yan, Yan Chen, Tiansheng Yi, Meisheng Ni, Hong Naruse, Kiyoshi Nagahama, Yoshitaka Hong, Yunhan |
author_facet | Guan, Guijun Yan, Yan Chen, Tiansheng Yi, Meisheng Ni, Hong Naruse, Kiyoshi Nagahama, Yoshitaka Hong, Yunhan |
author_sort | Guan, Guijun |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3654493 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Ivyspring International Publisher |
record_format | MEDLINE/PubMed |
spelling | pubmed-36544932013-05-15 Nanos3 Gene Targeting in Medaka ES Cells Guan, Guijun Yan, Yan Chen, Tiansheng Yi, Meisheng Ni, Hong Naruse, Kiyoshi Nagahama, Yoshitaka Hong, Yunhan Int J Biol Sci Research Paper 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. Ivyspring International Publisher 2013-05-09 /pmc/articles/PMC3654493/ /pubmed/23678294 http://dx.doi.org/10.7150/ijbs.6507 Text en © Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. |
spellingShingle | Research Paper Guan, Guijun Yan, Yan Chen, Tiansheng Yi, Meisheng Ni, Hong Naruse, Kiyoshi Nagahama, Yoshitaka Hong, Yunhan Nanos3 Gene Targeting in Medaka ES Cells |
title | Nanos3 Gene Targeting in Medaka ES Cells |
title_full | Nanos3 Gene Targeting in Medaka ES Cells |
title_fullStr | Nanos3 Gene Targeting in Medaka ES Cells |
title_full_unstemmed | Nanos3 Gene Targeting in Medaka ES Cells |
title_short | Nanos3 Gene Targeting in Medaka ES Cells |
title_sort | nanos3 gene targeting in medaka es cells |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654493/ https://www.ncbi.nlm.nih.gov/pubmed/23678294 http://dx.doi.org/10.7150/ijbs.6507 |
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