Light Activated Recombination

Abstract

Many genes elicit their actions through their expression in precise spatial patterns in tissues. Photoregulated expression systems offer a means to remotely pattern gene expression in tissues. Using currently available photopatterning methods, gene expression is only transient. Herein is described a general method to permanently alter a cell's genome under the control of light. The photocaged estrogen receptor (ER) antagonists, nitroveratryl-hydroxytamoxifen (Nv-HTam) and nitroveratryl-hydroxytamoxifen aziridine (Nv-HTaz), mediate exposure-dependent recombination in cells expressing the Cre-ER, a fusion of the site-specific recombinase Cre and ER. Both Nv-HTam and Nv-HTaz only activate recombination by Cre-ER after exposure to light. When released only intracellularly, the covalent-modifying Taz can mediate significant amounts of recombination in an exposure-dependent manner. Nv-HTaz and Cre-ER represent perhaps the first compound that can be used to photopattern gene expression through recombination.

Optochemogenetics (OCG) Allows More Precise Control of Genetic Engineering in Mice with CreER regulators

“…While caged 4-OHT analogues were shown to control CreER-mediated gene expression in response to photoactivation in cell culture 12 and zebrafish, 14, 15 our results demonstrate this approach can indeed be applied to adult mice to induce localized gene activation as a function of light illumination.…”

4-Hydroxytamoxifen probes for light-dependent spatiotemporal control of Cre-ER mediated reporter gene expression

“…[17][18][19][20][21] We previously published a study validating this concept using a photocaged tamoxifen (TAM). 22 In that study, an o-nitrobenzyl (ONB)-alkylated N-quaternary salt 15,23,24 of tamoxifen (ONB-TAM) was synthesized and found to be water soluble, and its activity was neutralized until exposed to long wave ultraviolet light (UVA, 365 nm).…”

Taking control of gene expression with light-activated oligonucleotides

“…These include diverse methods for focally delivering genetic material, such as by electro- or laser poration (10); the use of promoters with known temporal and tissue-specific expression profiles; diffusion of bioactive small molecules, which are typically obtained by screening large compound libraries (11); tetracycline-inducible constructs (12); Cre- lox P recombinase, which can be made inducible by heat-shock (13) or small molecules such as doxycycline, aldo-sterone, and tamoxifen (14); and light-activated (caged) compounds (3). We address the last method here, of which there are many examples: caged neurotransmitters (15,16), hormones (17,18), amino acids (1922), peptides (23,24), proteins (23,2532), calcium (33) and other second messengers such as cAMP (3437), and nitric oxide (NO) (38,39). In the caged state, these molecules are transiently blocked from performing their biological functions.…”