p53 Gene Targeting by Homologous Recombination in Fish ES Cells
| العنوان: | p53 Gene Targeting by Homologous Recombination in Fish ES Cells |
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| المؤلفون: | Ni Hong, Mingyou Li, Songlin Chen, Yan Yan, Yongkang Qiao, Guijun Guan, Tiansu Wang, Chang Ming Li, Yunhan Hong, Tiansheng Chen, Manfred Schartl |
| المساهمون: | School of Chemical and Biomedical Engineering |
| المصدر: | PLoS ONE PLoS ONE, Vol 8, Iss 3, p e59400 (2013) |
| سنة النشر: | 2013 |
| مصطلحات موضوعية: | Genotype, DNA recombination, Science, Oryzias, Animal Types, Genetic Vectors, Biochemistry, Model Organisms, ddc:571, Genetics, Animals, Site-specific recombinase technology, Homologous Recombination, Small Animals, Zebrafish, Gene, Biology, Embryonic Stem Cells, In Situ Hybridization, Multidisciplinary, biology, Stem Cells, Gene targeting, Transfection, DNA, Animal Models, biology.organism_classification, Genes, p53, Science::Biological sciences::Microbiology::Microorganisms [DRNTU], Embryonic stem cell, Nucleic acids, Blotting, Southern, Gene Targeting, Models, Animal, Medicine, Veterinary Science, Gene Function, Homologous recombination, Genetic Engineering, DNA modification, Research Article, Biotechnology, Transgenics, Developmental Biology |
| الوصف: | Background: Gene 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 similar to MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by similar to 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. |
| وصف الملف: | application/pdf |
| اللغة: | English |
| DOI: | 10.1371/journal.pone.0059400 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::db8403e133c633adb19d894018947836 https://doi.org/10.1371/journal.pone.0059400 |
| Rights: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....db8403e133c633adb19d894018947836 |
| قاعدة البيانات: | OpenAIRE |
| DOI: | 10.1371/journal.pone.0059400 |
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