Inactivation of an integrated antibiotic resistance gene in mammalian cells to re-enable antibiotic selection. (April 2014)

• Record Type: Journal Article
• Authors: Ni, Peiling; Zhang, Qian; Chen, Haixia; Chen, Lingyi
• Is Part Of: Biotechniques. Volume 56:Number 4(2014)
• Page Start: 198
• Page End: 201
• Publication Date: 2014-04
• Subjects: antibiotic resistance gene -- genetic engineering -- CRISPR/Cas; Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/000114160
• Languages: English
• ISSNs: 0736-6205

Abstract

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.