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|Title:||Baculoviral transduction facilitates TALEN-mediated targeted transgene integration and Cre/LoxP cassette exchange in human-induced pluripotent stem cells|
|Citation:||Zhu, H., Lau, C.-H., Goh, S.-L., Liang, Q., Chen, C., Du, S., Phang, R.-Z., Tay, F.C., Tan, W.-K., Li, Z., Tay, J.C.-K., Fan, W., Wang, S. (2013-10). Baculoviral transduction facilitates TALEN-mediated targeted transgene integration and Cre/LoxP cassette exchange in human-induced pluripotent stem cells. Nucleic Acids Research 41 (19) : e180-. ScholarBank@NUS Repository. https://doi.org/10.1093/nar/gkt721|
|Abstract:||Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells. © 2013 The Author(s) 2013.|
|Source Title:||Nucleic Acids Research|
|Appears in Collections:||Staff Publications|
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