Repetitive short‐pulsed illumination efficiently activates photoactivatable‐Cre as continuous illumination in embryonic stem cells and pre‐implantation embryos of transgenic mouse

Abstract

Summary The Cre‐loxP system has been widely used for specific DNA recombination which induces gene inactivation or expression. Recently, photoactivatable‐Cre (PA‐Cre) proteins have been developed as a tool for spatiotemporal control of the enzymatic activity of Cre recombinase. Here, we generated transgenic mice bearing a PA‐Cre gene and systematically investigated the conditions of photoactivation for the PA‐Cre in embryonic stem cells (ESCs) derived from the transgenic mice and in a simple mathematical model. Cre‐mediated DNA recombination was induced in 16% of the PA‐Cre ESCs by 6 hr continuous illumination. We show that repetitive pulsed illumination efficiently induced DNA recombination with low light energy as efficient as continuous illumination in the ESCs (96 ± 15% of continuous illumination when pulse cycle was 2 s), which was also supported by a minimal mathematical model. DNA recombination by the PA‐Cre was also successfully induced in the transgenic mouse pre‐implantation embryos under the developed conditions. These results suggest that strategies based on repetitive pulsed illumination are efficient for the activation of photoactivatable Cre and, possibly other photo‐switchable proteins.

Using a modified piggyBac transposon‐combined Cre/loxP system to produce selectable reporter‐free transgenic bovine mammary epithelial cells for somatic cell nuclear transfer

“…According to this report, the function of the Cre/loxP system was confirmed to be normal (Figure S2). Recent studies have also shown that selective marker genes can be successfully removed from transgenic mammalian cells by the Cre/loxP system (Bittel et al, 2021; Kishi et al, 2021). Considering that Cre recombinase was isolated from Coliphage P1 virus (Sauer & Henderson, 1988) and combining previous reports (Nolden et al, 2006b; Peitz, Pfannkuche, Rajewsky, & Edenhofer, 2002), using prokaryotic expression to produce a cell‐permeable NLS‐TAT‐Cre recombinase may retain its activity.…”