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PLoS One. 2018 Nov 29;13(11):e0201683. doi: 10.1371/journal.pone.0201683. eCollection 2018.

Highly efficient scarless knock-in of reporter genes into human and mouse pluripotent stem cells via transient antibiotic selection.

Author information

1
Department of Ophthalmology, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States of America.
2
Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
3
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine Baltimore, Maryland, United States of America.
4
Department of Molecular Biology and Genetics, The Solomon H. Snyder Department of Neuroscience, Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

Abstract

Pluripotent stem cells (PSCs) edited with genetic reporters are useful tools for differentiation analysis and for isolation of specific cell populations for study. Reporter integration into the genome is now commonly achieved by targeted DNA nuclease-enhanced homology directed repair (HDR). However, human PSCs are known to have a low frequency of gene knock-in (KI) by HDR, making reporter line generation an arduous process. Here, we report a methodology for scarless KI of large fluorescent reporter genes into PSCs by transient selection with puromycin or zeocin. With this method, we can perform targeted KI of a single reporter gene with up to 65% efficiency, as well as simultaneous KI of two reporter genes into different loci with up to 11% efficiency. Additionally, we demonstrate that this method also works in mouse PSCs.

PMID:
30496180
DOI:
10.1371/journal.pone.0201683
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Conflict of interest statement

We have read the journal’s policy and the authors of this manuscript have the following competing interests: V.M. Sluch, D.S. Rice Novartis Institutes for BioMedical Research, employment; X. Chamling, C. Wenger, Y. Duan, and D.J. Zack declare no competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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