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

Abstract

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 self‐cleaved inducible CreER (sCreER) 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 VEGFR2flox/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.

Expression of a tetracycline-controlled transactivator (Tet-On/Off system) in beta cells reduces insulin expression and secretion in mice

“…Novel tools are continuously being developed. Self-cleaved inducible CreER (sCreER) may help to increase efficiency of recombination 60 or a photoactivatable Cre 61 could non-invasively and temporally activate transgene expression without the need for drugs. Of course, with new technology comes new caveats that will themselves require characterization and their own unique controls.…”

Strategies for site-specific recombination with high efficiency and precise spatiotemporal resolution

“…Even if the cells with recombination are of a limited amount, sCreER switch into Cre can be detected by reporter. A combination of temporal control and constitutive Cre activity makes sCreER suitable for lineage tracing and gene deletion simultaneously ( 2 ). Many studies have displayed that expression of a given Cre-loxP recombination reporter cannot reliably indicate deletion of target gene.…”

“…Finally, novel strategies are required to induce gene expression and deletion regionally and promptly. All of these limitations could be circumvented with remarkable improvements and progress of SSRs technologies ( 2 , 3 , 4 ).…”

“… Efficient and temporally controlled gene deletion for functional study. ( 2 ) iSuRe-Cre Cross w/other Cre/CreER lines; w/or w/o tamoxifen induction High efficiency; temporal control; nontoxicity; no leakiness; reliably reports cells with gene deletion; compatible with the numerous existent loxP and Cre/CreERT2 alleles Does not prevent the occurrence of gene-deletion false-negatives Increasing the efficiency and reliability of Cre-dependent reporter and gene function analysis. ( 3 ) Di-Cre Rapamycin induction Tight temporal control; rapid induction; low background Cre activity Toxic during development; only can be used in adult animals Tight temporal control of recombinase activity for conditional gene knock-out.…”

Genetic Tools to Study Cardiovascular Biology

“…An alternative method presented recently to increase Cre activity after tamoxifen induction of CreERT uses an inducible self-cleaved CreER (sCreER), in which the ER element is flanked by LoxP sites. In the non-induced state, Cre-LoxP-ER-LoxP is expressed but inactive, and after tamoxifen the ER portion is deleted in the induced cells, and Cre is free to mobilize to the nucleus and recombine other flanked genes (Tian et al, 2020). This strategy boosts Cre expression and activity in initially CreER recombined cells; however, the sCreER lines generated so far are tissue-specific and also do not contain an internal reporter linked with Cre expression, in order to ensure that cells expressing an independent Cre reporter have full gene deletion.…”