Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition.
Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition.
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DOI：
10.1016/j.stemcr.2015.09.004
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发表时间：
2015-11-10
影响因子：
5.9
通讯作者：
Verfaillie CM
中科院分区：
文献类型：
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作者：
Ordovás L;Boon R;Pistoni M;Chen Y;Wolfs E;Guo W;Sambathkumar R;Bobis-Wozowicz S;Helsen N;Vanhove J;Berckmans P;Cai Q;Vanuytsel K;Eggermont K;Vanslembrouck V;Schmidt BZ;Raitano S;Van Den Bosch L;Nahmias Y;Cathomen T;Struys T;Verfaillie CM
在同基因背景下“安全港”基因座快速高效转基因的工具对于开发人类多能干细胞（hPSC）的可能性仍然很重要。我们在 AAVS1 基因座中创建了适合 FLPe 重组酶介导的盒交换 (RMCE) 的 hPSC 主细胞系，可在 15 天内以 100% 的效率生成转基因细胞系，且无需随机整合。使用 RMCE，我们成功地整合了几种可用于谱系鉴定、细胞毒性研究和基因过表达以研究肝细胞谱系的转基因。然而，由于 DNA 甲基化和其他未知机制，我们在未分化的 hESC 和分化的肝细胞中观察到了体外意想不到的、可变的转基因表达抑制。因此，AAVS1 基因座不能被认为是体外可靠转基因表达的普遍安全港基因座，并且将其用于 hPSC 转基因需要仔细评估各个转基因的功能。使用正/负选择的 RMCE 允许在 15 天内生成转基因 hPSC 系 AAVS1 基因座通过诱导从头 DNA 甲基化发挥转基因抑制作用 AAVS1 介导的沉默在体外出现序列和谱系特异性，但在体内不存在 AAVS1 不符合被视为体外通用安全港基因座的要求 Verfaillie、Ordovás 及其同事 报告了一种高效、快速的方法，可以在 AAVS1 安全港基因座中对 hPSC 进行基因改造，而无需发生随机整合事件。他们的结果证明了该基因座适用于研究肝谱系的多种应用，但他们发现了未分化和肝细胞分化 hESC 中迄今为止未知的可变 AAVS1 介导的转基因抑制。因此，AAVS1 不能被视为安全港基因座。
Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes. RMCE using positive/negative selection allows generation of transgenic hPSC lines in 15 days The AAVS1 locus exerts transgene inhibition by inducing de novo DNA methylation AAVS1-mediated silencing appears sequence and lineage-specific in vitro, yet is not present in vivo AAVS1 does not meet the requirements to be considered a universal safe harbor locus in vitro Verfaillie, Ordovás, and colleagues report an efficient and fast method to genetically engineer hPSC in the AAVS1 safe harbor locus free of random integration events. Their results demonstrate the suitability of the locus for several applications to study the hepatic lineage, but they uncover a so-far-unknown variable AAVS1-mediated transgene inhibition in undifferentiated and hepatocyte differentiating hESC. Therefore, AAVS1 cannot be considered a safe harbor locus.