Light-Activated Gene Expression Directs Segregation of Co-cultured Cells in Vitro

Abstract

Light-directed gene patterning methods have been described as a means to regulate gene expression in a spatially and temporally controlled manner. Several methods have been reported that use photocaged forms of small molecule effectors to control ligand-dependent transcription factors. Whereas these methods offer many advantages including high specificity and transient light-sensitivity, the free diffusion of the uncaged effector can limit both the magnitude and resolution of localized gene induction. Methods to date have been limited by the small fraction of irradiated cells that have expression levels significantly above uninduced background and have not been shown to affect a defined biological response. The tetracycline-dependent transactivator/transrepressor system, RetroTET-ART, combined with a photocaged form of doxycycline (NvOC-Dox) can be used to form photolithographic patterns of induced expression wherein up to 85% of the patterned cells show expression levels above uninduced regions. The efficiency and inducibility of the RetroTET-ART system allows one to quantitatively measure the limits of resolution and the relative induction levels mediated by a small molecule photocaged effector for the first time. Well-defined patterns of reporter genes were reproducibly formed within 6-36 h with feature sizes as small as 300 microm. After photo-patterning, NvOC-Dox can be rapidly removed, rendering cells photoinsensitive and allowing one to monitor GFP product formation in real time. Patterned co-expression of the cell surface ligand ephrin A5 on cell monolayers creates well-defined patterns that are sufficient to direct and segregate co-cultured cells via either attractive or repulsive signaling cues. The ability to direct the arrangement of cells on living cell monolayers through the action of light may serve as a model system for engineering artificial tissues.

Three-dimensional patterning of multiple cell populations through orthogonal genetic control of cell motility

“…75, 76 One could also utilize caged variants of doxycycline that can be irreversibly uncaged with UV light to stimulate doxycycline-inducible gene expression in specific cells. 77, 78 This method has been used to create cell patterns both in 3D tissue extracts and in vivo using two-photon microscopy. 79 To impose additional control over the direction of cell migration, one could use materials engineering techniques, such as soft lithography, photolithography or 3D printing, to introduce physical cues into the ECM.…”

An Engineered Optogenetic Switch for Spatiotemporal Control of Gene Expression, Cell Differentiation, and Tissue Morphogenesis

“…This contrasts to other genetic switches, such as the tetracycline-inducible system 47, 48 , that require the addition of a small molecule or hormone 49, 50 to activate gene expression. Photocaged unnatural amino acids or chemical inducers have also enabled a variety of approaches to inducible transcription and recombination controlled by UV light 12, 13, 51–54 . However, these methods currently require partial amber stop codon suppression to direct the incorporation of the photoreactive residue 55 .…”

Recent advances in the photochemical control of protein function

“…More defined spatial control over protein expression was recently demonstrated in mammalian cells using a caged doxycycline molecule 2 (Figure 2c; the caging group is shown in red)19. The doxycycline-inducible gene expression system RetroTET-Art (containing a doxycycline-reactive repressor/activator pair)20 was employed in conjunction with the caged molecule 2 19.…”