Open AccessHighly efficient scarless knock-in of reporter genes into human and mouse pluripotent stem cells via transient antibiotic selection
Author(s) -
Valentin M. Sluch,
Xitiz Chamling,
Claire Wenger,
Yun-you Duan,
Dennis S. Rice,
Donald J. Zack
Publication year - 2018
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0201683
Subject(s) - reporter gene , induced pluripotent stem cell , biology , gene , gene knockin , gene targeting , puromycin , gene knockout , hek 293 cells , homologous recombination , embryonic stem cell , microbiology and biotechnology , computational biology , genetics , gene expression , protein biosynthesis
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.