Open AccessInactivation of an integrated antibiotic resistance gene in mammalian cells to re-enable antibiotic selection
Author(s) -
Peiling Ni,
Qian Zhang,
Hạixia Chen,
Lingyi Chen
Publication year - 2014
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000114160
Subject(s) - puromycin , crispr , gene , biology , embryonic stem cell , antibiotics , antibiotic resistance , reporter gene , gene targeting , cell culture , genetics , microbiology and biotechnology , gene expression , protein biosynthesis
Removing an antibiotic resistance gene allows the same antibiotic to be re-used in the next round of genetic manipulation. Here we applied the CRISPR/Cas system to disrupt the puromycin resistance gene in an engineered mouse embryonic stem cell line and then re-used puromycin selection in the resulting cells to establish stable reporter cell lines. With the CRISPR/Cas system, pre-engineered sequences, such as loxP or FRT, are not required. Thus, this technique can be used to disrupt antibiotic resistance genes that cannot be removed by the Cre-loxP and Flp-FRT systems.