Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats
Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats
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DOI：
10.1007/s11248-011-9564-0
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发表时间：
2012-08-01
影响因子：
3
通讯作者：
Hashimoto, Tadatoshi
中科院分区：
文献类型：
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作者：
Yamamoto, Satoshi;Nakata, Mitsugu;Hashimoto, Tadatoshi
2008-2010年期间报道的基因敲除（KO）大鼠生产的显著成就之一是使用含有糖原合酶激酶3和促分裂原活化蛋白激酶激酶抑制剂的新型培养基（2 i培养基）从大鼠胚泡衍生真实的胚胎干（ES）细胞。在这里，我们报告基因靶向技术通过同源重组在大鼠ES细胞，证明其使用通过生产蛋白酶激活受体-2基因（Par-2）KO大鼠。我们首先使用2 i培养基从Dark Agglomerate大鼠中产生生殖系感受态ES细胞。这些ES细胞在体外分化为心肌细胞，当注射到囊胚中时可以产生具有高ES细胞贡献的嵌合体。然后，我们引入了具有由CAG启动子驱动的新霉素抗性基因的靶向载体来破坏Par-2。经过7天的药物筛选，获得489个新霉素抗性菌落。通过聚合酶链反应（PCR）基因分型和定量PCR分析筛选后，我们确认了三个同源重组克隆，产生了将Par-2靶向等位基因传递给后代的嵌合体。Par-2 KO大鼠胃细胞中Par-2信使RNA表达缺失，主动脉中缺少PAR-2介导的平滑肌松弛，如药理学试验所示。与小鼠相比，大鼠在生物医学研究中具有许多优势，包括更大的体型;因此，它们被广泛用于科学研究。因此，利用大鼠ES细胞建立基因打靶技术将是人类疾病模型生产和药物发现的有价值的工具。
One of the remarkable achievements in knockout (KO) rat production reported during the period 2008-2010 is the derivation of authentic embryonic stem (ES) cells from rat blastocysts using a novel culture medium containing glycogen synthase kinase 3 and mitogen-activated protein kinase kinase inhibitors (2i medium). Here, we report gene-targeting technology via homologous recombination in rat ES cells, demonstrating its use through production of a protease-activated receptor-2 gene (Par-2) KO rat. We began by generating germline-competent ES cells from Dark Agouti rats using 2i medium. These ES cells, which differentiate into cardiomyocytes in vitro, can produce chimeras with high ES cell contribution when injected into blastocysts. We then introduced a targeting vector with a neomycin-resistant gene driven by the CAG promoter to disrupt Par-2. After a 7-day drug selection, 489 neomycin-resistant colonies were obtained. Following screening by polymerase chain reaction (PCR) genotyping and quantitative PCR analysis, we confirmed three homologous recombinant clones, resulting in chimeras that transmitted the Par-2 targeted allele to offspring. Par-2 KO rats showed a loss of Par-2 messenger RNA expression in their stomach cells and a lack of PAR-2 mediated smooth muscle relaxation in the aorta as indicated by pharmacological testing. Compared with mice, rats offer many advantages in biomedical research, including a larger body size; consequently, they are widely used in scientific investigation. Thus, the establishment of a gene-targeting technology using rat ES cells will be a valuable tool in human disease model production and drug discovery.