CreERT2 vector and pcDNA3.1 empty vector (Mock) treated with 1 µM tamoxifen (Tam) addition 36 h after the transfection were used as positive and negative controls, respectively (* P < 0.05 **** P < 0.0001 dark v.s. The herpes simplex virus thymedine kinase (HSV-TK) promoter- Renilla Luc plasmid was co-transfected as a transfection control to normalize Firefly Luc activity. Luc assays were conducted with double-floxed inverted Fluc reporter in HEK 293T cells. Upper diagram shows experimental protocol used for Luc assay (blue LED, 447.5 nm, 8.28 W/m 2, repeated 20 s light and 60 s dark for 12 h). b Comparison of PA-Cre with various blue-light photoreceptors using luciferase (Luc) assay. Split Cre (59/60) are complemented along with nMag–pMag dimerization upon blue-light illumination (BL blue light, NLS nuclear localization signal, 2A P2A self-cleaving peptide sequence, P CMV cytomegalovirus promoter, Fluc Firefly luciferase, pA polyadenylation transcriptional “stop” (poly-A) signal repeated sequence). To address this issue, we looked to improve the Magnets-based PA-Cre system by reducing the background dark activity.Ĭomparison of multiple light-activated dimerization systems in photoactivatable Cre recombinase.Ī Schematic representation of photoactivatable (PA)-Cre system and its reporter constructs. Such leaky recombination in dark after PA-Cre expression is not acceptable for any in vivo applications as Cre- loxP recombination is irreversible. The Magnets-based PA-Cre showed an accumulating leak over time while the CRY2/CIB1 version demonstrated little to no leakiness (Supplementary Fig. 11), was also tested as a benchmark experiment. The CRY2/CIB1-based construct, called PA-Cre 2.0 (ref. To assess this dark leakiness issue further, we monitored the Luc activity 24, 48, 72, and 96 h after HEK 293T cells were transfected with the PA-Cre constructs. These results suggest that the original Magnets-based PA-Cre is still promising for further improvement as the unintentional Cre- loxP dark leak recombination is limited (Supplementary Fig. On the other hand, the iLID/SspB-based version had much higher leakiness in dark than the others. The FKF1/GI-based version also demonstrated as low Cre- loxP recombination efficiency with blue light as the CRY2/CIB1-based one (called PA-Cre 2.0) 11. While the fold induction of Cre- loxP recombination using the CRY2/CIB1-based PA-Cre is the best (43.3×) among the tests, the efficiency of Cre- loxP recombination with light was low (~15%) compared with CreERT2 positive control. We found that the Magnets-based original PA-Cre had the highest Cre- loxP recombination efficiency with light among these constructs (~75% compared with a positive control, CreERT2, treated with tamoxifen). To address this question, CRY2/CIB1-, iLID/SspB-, and FKF1/GI-based PA-Cre constructs were prepared and tested using luciferase (Luc) and mCherry reporters, and compared to the original Magnets-based PA-Cre 9, 11– 13 (Fig. Although the construct of PA-Cre could be successfully transiently applied in mammalian cells in vitro and mouse livers in vivo using hydrodynamic tail vein (HTV) injection and reporter plasmids, other blue-light-inducible hetero-dimerization systems could be more suitable for developing PA-Cre systems. Previously, we developed the 1st generation of Magnets-based PA-Cre, taking advantage of blue-light-dependent hetero-dimerization system, Magnets 10. We believe this improved system and mouse model availability can enhance genetic studies in living systems to address biological hypotheses and unveil the molecular and pathophysiological mechanisms underlying various diseases. We demonstrate the improved efficiency of PA-Cre 3.0 and its applications in vivo using newly generated mouse lines expressing PA-Cre 3.0 conditionally. In this study, we developed an improved version of PA-Cre called PA-Cre 3.0, which is based on the same blue-light-dependent dimerization system, Magnets. In addition, there are currently no in vivo mouse models available for optogenetic-based systems, limiting the scope of applications in biological study. While our previously reported Magnets-based PA-Cre system improved on many of these shortcomings 10, PA-Cre still had a major issue with unintentional recombination in dark conditions prior to light stimulation. While various inducible systems have been developed based on Cre- loxP recombination, the tools often suffer from either low efficiency (such as with the CRY2-CIB1-based system) or have complications such as the necessity for harmful chemical inducers such as tamoxifen or rapamycin 6– 9. Cre recombinase, which is derived from P1 bacteriophage, is the most common recombinase that has been used to catalyze directional DNA recombination between loxP pairs 2– 5. The ability to manipulate DNA recombination and gene expression in a spatiotemporal specific manner is a powerful technique in genome engineering studies 1.
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