An optimized proliferation system of embryonic stem cells for generating the rat model with large fragment modification
An optimized proliferation system of embryonic stem cells for generating the rat model with large fragment modification
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一种优化的胚胎干细胞增殖体系，用于产生大片段修饰的大鼠模型
DOI：
10.1016/j.bbrc.2021.07.053
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发表时间：
2021
影响因子：
3.1
通讯作者：
Peng Luying
中科院分区：
文献类型：
--
作者：
Liu Chang;Cui Zhonglin;Yan Youzhen;Wu Nancy L.;Li Li;Ying Qilong;Peng Luying
长期以来，大鼠一直是生物医学领域疾病研究的理想模型，但大鼠胚胎干细胞体外培养的瓶颈阻碍了其作为遗传病模型的广泛应用。在此，我们对一种特殊的大鼠胚胎干细胞培养液进行了优化，命名为5N-培养液，使体外培养的细胞具有更好的形态和更高的多能性。然后，我们建立了一个药物选择时间表，其中预先选择了12小时，实现了更高的阳性选择率。与常规的2I培养条件相比，这些处理使同源重组效率至少提高了50%。此外，整倍体ES克隆的比例也提高了50%，生殖系传播率更高。最后，我们通过重组酶介导盒交换(RMCE)成功地将175kb的人细菌人工染色体(BAC)片段插入到大鼠的ES基因组中。因此，我们提供了一个很有前途的系统来建立复杂的大鼠模型，这可能有助于生物医学研究。
Rats have long been an ideal model for disease research in the field of biomedicine, but the bottleneck of in vitro culture of rat embryonic stem (ES) cells hindered the wide application as genetic disease models. Here, we optimized a special medium which we named 5N-medium for rat embryonic stem cells, which improved the in vitro cells with better morphology and higher pluripotency. We then established a drug selection schedule harboring a prior selection of 12 h that achieved a higher positive selection ratio. These treatments induced at least 50% increase of homologous recombination efficiency compared with conventional 2i culture condition. Moreover, the ratio of euploid ES clones also increased by 50% with a higher germline transmission rate. Finally, we successfully knocked in a 175 kb human Bacterial Artificial Chromosome (BAC) fragment to rat ES genome through recombinase mediated cassette exchange (RMCE). Hence, we provide a promising system for generating sophisticated rat models which could be benefit for biomedical researches.