Generation of a self‐cleaved inducible Cre recombinase for efficient temporal genetic manipulation

Site‐specific recombinase‐mediated genetic technology, such as inducible Cre‐loxP recombination (CreER), is widely used for in vivo genetic manipulation with temporal control. The Cre‐loxP technology improves our understanding on the in vivo function of specific genes in organ development, tissue regeneration, and disease progression. However, inducible CreER often remains inefficient in gene deletion. In order to improve the efficiency of gene manipulation, we generated a s elf‐cleaved inducible CreER ( s CreER) that switches inducible CreER into a constitutively active Cre by itself. We generated endocardial driver Npr3‐sCreER and fibroblast driver Col1a2‐sCreER , and compared them with conventional Npr3‐CreER and Col1a2‐CreER, respectively. For easy‐to‐recombine alleles such as R26‐tdTomato , there was no significant difference in recombination efficiency between sCreER and the conventional CreER. However, for alleles that were relatively inert for recombination such as R26‐Confetti , R26‐LZLT , R26‐GFP , or VEGFR2 flox / flox alleles, sCreER showed a significantly higher efficiency in recombination compared with conventional CreER in endocardial cells or fibroblasts. Compared with conventional CreER, sCreER significantly enhances the efficiency of recombination to induce gene expression or gene deletion, allowing temporal yet effective in vivo genomic modification for studying gene function in specific cell lineages. Synopsis A self‐cleaved CreER (sCreER) is generated by placing two loxP sites on the flanks of ER cassette. After tamoxifen (Tam) induction, sCreER protein is transported into the nucleus where it cleaves loxP flanked ER, converting sCreER DNA into Cre genotype. Thus, the Cre recombinase can be switched from an inducible form to a constitutively active form. sCreER significantly enhances the efficiency of gene manipulation on some alleles that are relatively inert for recombination. sCreER exhibits temporal control property by tamoxifen. sCreER self‐cleaves and converts to Cre for robust gene deletion. sCreER is more efficient than CreER in recombination of a refractory or less susceptible target locus. Graphical Abstract Methodological development of the inducible Cre recombinase system for in vivo genetic manipulation, based on self‐conversion of an inducible to a constitutively active Cre form, results in higher recombination efficiency than conventional Cre‐based systems.