Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
Stem Cell Reports. 2015 Nov 10;5(5):918-931. doi: 10.1016/j.stemcr.2015.09.004. Epub 2015 Oct 8.
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.
在同基因背景下，用于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。我们创建了 hPSC 主细胞系，适合在 AAVS1 基因座中进行 FLPe 重组酶介导的盒交换（RMCE），这使得在 15 天内以 100%的效率生成转基因系，并且没有随机整合。使用 RMCE，我们成功地整合了几个用于谱系鉴定、细胞毒性研究和基因过表达的转基因，以研究肝谱系。然而，我们在体外观察到意想不到的和可变的转基因表达抑制，这是由于 DNA 甲基化和其他未知机制，无论是在未分化的 hESC 还是分化的肝细胞中。因此，AAVS1 基因座不能被视为体外可靠转基因表达的通用安全港基因座，并且在 hPSC 中使用它进行转基因将需要仔细评估单个转基因的功能。
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