Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus
Resumen: Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.
Idioma: Inglés
DOI: 10.3791/54718
Año: 2016
Publicado en: Journal of visualized experiments : JoVE 117 (2016), [11 pp.]
ISSN: 1940-087X

Factor impacto JCR: 1.232 (2016)
Categ. JCR: MULTIDISCIPLINARY SCIENCES rank: 27 / 63 = 0.429 (2016) - Q2 - T2
Factor impacto SCIMAGO: 0.866 - Chemical Engineering (miscellaneous) (Q1) - Immunology and Microbiology (miscellaneous) (Q2) - Biochemistry, Genetics and Molecular Biology (miscellaneous) (Q2) - Neuroscience (miscellaneous) (Q3)

Tipo y forma: Article (Published version)

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