Transfection of mammalian cells with exogenous DNA such as plasmids is a valuable technique for exploring the biological functions of a gene of interest. Traditional gene delivery approaches using non-viral vectors employ drug resistance genes as selectable markers, leaving a genetic ‘scar’ that can interfere with cell survival and with the analysis of the resulting phenotype. Furthermore, it is difficult to perform gene transfer in multiple-drug resistant cells. Fluorescence-activated cell sorting (FACS) is a powerful method for enrichment of transfected cells under drug-free conditions, but the equipment used for FACS is very expensive and sometimes laborious to operate. Therefore, novel methods are required for obtaining stable transfectants that do not depend on drug selection and can be performed in a more convenient and easier manner. In this review, we summarize the achievements of the drug-free acquisition of gene-engineered mammalian cells. Special attention is focused on the recent advances in the acquisition of mutated cells by using CRISPR/Cas9-based genome editing, a novel and recently developed technology, under drug-free conditions.
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