CIRKO: A chemical-induced reversible gene knockout system for studying gene function in situ

Conditional loss and restoration of function are becoming important approaches for investigating gene function. Given that reversible conditional gene knockouts in cells required complicated manipulation, conditional inactivation and reactivation of a gene in primary somatic cells with limited proliferative capacity and in animal models remain difficult to achieve. Here, we first developed a reportable and reversible conditional intronic cassette (ReCOIN), wherein inactivation and reactivation of the gene are mediated via sequential expression of Cre and Flp recombinases, respectively. The expression pattern of the target gene can be monitored by direct visualization. To simply and tightly control temporal expression of the recombinases, on the basis of ReCOIN, we further presented a dual chemical-induced reversible gene knockout system (CIRKO) by insertion of reverse tetracycline transcriptional activator (rtTA) and tetracycline response element (TRE)-controlled Cre and FlpoERT2 recombinases cassettes into Rosa26 and Hipp11 loci of cells, respectively, in which transcription termination of the target gene can be induced at a specific stage in the presence of doxycycline, while gene restoration is achieved in the presence of doxycycline and tamoxifen simultaneously. This system provides a simple, rapid, and flexible gene switch for studying gene function in situ both in vitro and in vivo.