dc.contributor.authorYan, Yan
dc.contributor.authorHong, Ni
dc.contributor.authorChen, Tiansheng
dc.contributor.authorLi, Mingyou
dc.contributor.authorWang, Tiansu
dc.contributor.authorGuan, Guijun
dc.contributor.authorQiao, Yongkang
dc.contributor.authorChen, Songlin
dc.contributor.authorSchartl, Manfred
dc.contributor.authorLi, Chang Ming
dc.contributor.authorHong, Yunhan
dc.date.accessioned2013-05-03T07:54:59Z
dc.date.available2013-05-03T07:54:59Z
dc.date.copyright2013en_US
dc.date.issued2013
dc.identifier.citationYan, Y., Hong, N., Chen, T., Li, M., Wang, T., Guan, G., Qiao, Y., Chen, S., Schartl, M., Li, C. M., & Hong, Y. (2013). p53 Gene Targeting by Homologous Recombination in Fish ES Cells. PLoS ONE, 8(3).en_US
dc.identifier.issn1932-6203en_US
dc.identifier.urihttp://hdl.handle.net/10220/9878
dc.description.abstractGene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes). Methodology and Principal Findings: Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1~MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by ~12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo. Conclusions: Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.en_US
dc.language.isoenen_US
dc.relation.ispartofseriesPLoS ONEen_US
dc.rights© 2013 The Author(s).en_US
dc.subjectDRNTU::Science::Biological sciences::Microbiology::Microorganisms
dc.titleP53 gene targeting by homologous recombination in fish ES cellsen_US
dc.typeJournal Article
dc.contributor.schoolSchool of Chemical and Biomedical Engineeringen_US
dc.identifier.doihttp://dx.doi.org/10.1371/journal.pone.0059400
dc.description.versionPublished versionen_US


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