Functional study of residual iCre activity relevant for split-Cre applications

Résumé fourni par la source

• Split-Cre recombinase system for controlling Cre recombinase activity. • Cre-mediated recombination functional assay relevant for Split-Cre strategies. • Identification of residual recombinase activity of C-terminal Cre protein fragment. Cre-lox system is a major tool in mouse molecular genetics instrumental in promoting somatic recombination to spatiotemporally control transcriptional activation/inhibition in subsets of cells or tissues in vivo . A critical factor behind this system may be represented by the availability of a specific promoter driving Cre expression in the cell subset of interest. Split-Cre recombinase system represents an evolution that circumvents this limitation using split N- and C-terminal domains of Cre recombinase placed under the control of two distinct promoters defining an intersectional domain where functional complementation of Cre protein fragments is obtained. This system is a valuable tool for controlling Cre recombinase activity in a spatially and temporally defined manner based on the assumption that neither N- or C-terminal Cre fragments alone have recombinase activity. However, residual recombinase activity of one of the two fragments can occur leading to confounding experimental results. In this work, we delve into characterizing functional activity of different N-terminal deleted codon-optimized Cre (iCre) isoforms to refine Split-Cre-based technologies, aiming to avoid uncontrolled recombinase events. Given the presence of several methionine residues in the amino acidic iCre sequence, we explored whether these residues could serve as potential translation start sites, resulting in truncated isoforms that might retain recombinase activity. To address this question, we tested in HEK293T cells whether site-specific recombination was retained in progressively amino-terminal deleted iCre isoforms. Our results reveal residual enzymatic activity of most amino-terminal deleted isoforms of iCre whose ATG start codon is located downstream of the commonly used split site. This insight holds significance for future refinements of the widely used Split-Cre system, providing information to avoid false positive results stemming from unwanted activity.

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