p53 gene targeting by homologous recombination in fish ES cells.
p53 gene targeting by homologous recombination in fish ES cells.
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DOI：
10.1371/journal.pone.0059400
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Hong Y
中科院分区：
文献类型：
--
作者：
Yan Y;Hong N;Chen T;Li M;Wang T;Guan G;Qiao Y;Chen S;Schartl M;Li CM;Hong Y
基因打靶技术为在胚胎干细胞中产生精确的遗传改变以阐明基因功能和建立人类疾病的动物模型提供了强有力的工具。然而，这项技术仅限于小鼠和大鼠。我们以前已经建立了ES细胞系和程序的基因转移和选择的同源重组（HR）事件在青鳉（Oryzias latipes）。在这里，我们报告HR介导的GT在这种生物体。我们设计了一个GT载体来破坏肿瘤抑制基因p53（也称为tp 53）。我们表明，所有三个青少年ES细胞系，MES 1和MES 3，是高度熟练的人力资源，因为他们产生可检测的人力资源没有药物选择。此外，阳性-阴性选择（PNS）程序使HR提高了1.12倍。在分析的39个PNS抗性菌落中，19个（48.7%）通过PCR基因分型为GT阳性。对11个PCR阳性克隆进行进一步分析，经Southern印迹分析、测序和荧光原位杂交，发现6个（54.5%）为真正的同源重组子。这在PNS条件下对p53 GT产生高达26.6%的高效率。我们表明，p53的破坏和长期繁殖的药物选择条件下不妥协的多能性，作为p53靶向的ES细胞保持稳定的生长，未分化的表型，多能性基因表达谱和分化潜力，在体外和体内。我们的研究结果表明，青鳉ES细胞是精通HR介导的GT，提供了第一个模式生物的低等脊椎动物的发展完全ES细胞为基础的GT技术。
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). 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. 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.
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影响因子：
4.5
作者：
Chen, SL;Hong, YH;Schartl, M
通讯作者：
Schartl, M
影响因子：
4
作者：
Hong, Ni;Chen, Songlin;Hong, Yunhan
通讯作者：
Hong, Yunhan
影响因子：
2.6
作者：
Li, Mingyou;Hong, Ni;Hong, Yunhan
通讯作者：
Hong, Yunhan
影响因子：
64.8
作者：
DONEHOWER, LA;HARVEY, M;BRADLEY, A
通讯作者：
BRADLEY, A
影响因子：
3
作者：
Hong, YH;Chen, SL;Schartl, M
通讯作者：
Schartl, M