000063497 001__ 63497
000063497 005__ 20200221144329.0
000063497 0247_ $$2doi$$a10.3791/54718
000063497 0248_ $$2sideral$$a103218
000063497 037__ $$aART-2016-103218
000063497 041__ $$aeng
000063497 100__ $$aOrdovás, L
000063497 245__ $$aRapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus
000063497 260__ $$c2016
000063497 5060_ $$aAccess copy available to the general public$$fUnrestricted
000063497 5203_ $$aEven 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.
000063497 540__ $$9info:eu-repo/semantics/openAccess$$aby-nc-nd$$uhttp://creativecommons.org/licenses/by-nc-nd/3.0/es/
000063497 590__ $$a1.232$$b2016
000063497 591__ $$aMULTIDISCIPLINARY SCIENCES$$b27 / 63 = 0.429$$c2016$$dQ2$$eT2
000063497 592__ $$a0.866$$b2016
000063497 593__ $$aChemical Engineering (miscellaneous)$$c2016$$dQ1
000063497 593__ $$aImmunology and Microbiology (miscellaneous)$$c2016$$dQ2
000063497 593__ $$aBiochemistry, Genetics and Molecular Biology (miscellaneous)$$c2016$$dQ2
000063497 593__ $$aNeuroscience (miscellaneous)$$c2016$$dQ3
000063497 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion
000063497 700__ $$aBoon, R
000063497 700__ $$aPistoni, M
000063497 700__ $$aChen, Y
000063497 700__ $$aSambathkumar, R
000063497 700__ $$aHelsen, N
000063497 700__ $$aVanhove, J
000063497 700__ $$aBerckmans, P
000063497 700__ $$aCai, Q
000063497 700__ $$aVanuytsel, K
000063497 700__ $$aRaitano, S
000063497 700__ $$aVerfaillie, CM
000063497 773__ $$g117 (2016), [11 pp.]$$pJ. vis. exp.$$tJournal of visualized experiments : JoVE$$x1940-087X
000063497 8564_ $$s416523$$uhttps://zaguan.unizar.es/record/63497/files/texto_completo.pdf$$yVersión publicada
000063497 8564_ $$s17253$$uhttps://zaguan.unizar.es/record/63497/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada
000063497 909CO $$ooai:zaguan.unizar.es:63497$$particulos$$pdriver
000063497 951__ $$a2020-02-21-13:45:11
000063497 980__ $$aARTICLE