Optochemogenetics (OCG) allows more precise control of genetic engineering in mice with CreER regulators

Abstract

New approaches that allow precise spatiotemporal control of gene expression in model organisms at the single cell level are necessary to better dissect the role of specific genes and cell populations in development, disease, and therapy. Here, we describe a new optochemogenetic switch (OCG switch) to control CreER/loxP-mediated recombination via photoactivatable ("caged") tamoxifen analogues in individual cells in cell culture, organoid culture, and in vivo in adult mice. This approach opens opportunities to more fully exploit existing CreER transgenic mouse strains to achieve more precise temporal- and location-specific regulation of genetic events and gene expression.

Figure 1

Schematics showing the photoactivation-dependent CreER/loxP system in the Rosa26CreER^T2;mT/mG reporter mice. To produce a light-sensitive CreER/loxP system, 4-OHC was modified with a light sensitive caging group to inhibit its ability to induce CreER-mediated recombination. Photoactivated release of 4-OHC induces EGFP gene expression in illuminated cells.

Figure 2

Synthesis of caged 4-OHC and photocleavage characterization. (A) Synthesis of caged 4-OHC under Mitsonobu reaction condition and activation (uncaging) of caged 4-OHC using 365 nm UV light. (B) Changes in UV-Vis absorbance during the photocleavage of caged 4-OHC in water:acetonitrile (1:1 v/v). Inset shows the change in absorbance at 375 nm over UV irradiation time. Note that the photocleavage is complete within 10 min of irradiation. (C) HPLC-MS chromatograms showing the quantitative formation 4-OHC from caged 4-OHC upon UV irradiation. Caged 4-OHC solutions, before (0 min) and after light exposure (10 min UV), were analyzed by HPLC-MS. Formation of 4-OHC was identified by the appearance of molecular mass corresponding to 4-OHC (m/z 352.31 [M+H]⁺). The peak other than the 4-OHC appeared in the 10 min UV chromatogram belongs to the cleaved caging group (see Figure 2A for the photocleavage reaction).

Figure 3

Photoactivation of caged 4-OHC in vitro. (A) Induction of EGFP expression by 365 nm UV activation of caged 4-OHC in MEFs isolated from Rosa26CreER^T2;mT/mG embryos. (B) Flow cytometric quantification of EGFP⁺ MEF cells upon treatment with 4-OHC and caged 4-OHC under different durations of UV activation. The ‘Intracellular + medium” group represents cell samples without PBS wash before UV irradiation, whereas the ‘Intracellular only’ group represents cell samples washed twice with PBS before UV irradiation. Data represent mean ± SD. (C) EGFP expression induced by 365 nm UV activation of caged 4-OHC in mammary epithelial cells isolated from Rosa26CreER^T2;mT/mG female mice and cultured to form acini on Matrigel. All acini grown on the dish were illuminated in a uniform manner to induce EGFP expression. Images shown are projections of the Z-stack confocal images.

Figure 4

Photoactivation of caged 4-OHC in vivo. (A) Strong induction in EGFP expression on the ventral skin as a result of 365 nm UV illumination of the Rosa26CreER^T2;mT/mG mice injected with caged 4-OHC. The dorsal skin was not illuminated and showed little EGFP signal. An increase in EGFP signal is followed by a decrease of tdTomato signal; low values of EGFP/tdTomato ratio correspond to low EGFP and high tdTomato, while high values of ratios correspond to high EGFP and low tdTomato. (B) Experimental design for intraperitoneal injection of vehicle or caged 4-OHC into the Rosa26CreER^T2;mT/mG female mice and subsequent 365 nm illumination on the right inguinal (#4) mammary fat pad. (C–D) The results of experiments in (B) with mammary tissue ex vivo imaged with IV-110. Only the right illuminated mammary gland from the mouse injected with caged 4-OHC expressed EGFP.