5 research outputs found
Light-Inducible Spatiotemporal Control of Gene Activation by Customizable Zinc Finger Transcription Factors
Advanced gene regulatory systems are necessary for scientific
research,
synthetic biology, and gene-based medicine. An ideal system would
allow facile spatiotemporal manipulation of gene expression within
a cell population that is tunable, reversible, repeatable, and can
be targeted to diverse DNA sequences. To meet these criteria, a gene
regulation system was engineered that combines light-sensitive proteins
and programmable zinc finger transcription factors. This system, light-inducible
transcription using engineered zinc finger proteins (LITEZ), uses
two light-inducible dimerizing proteins from <i>Arabidopsis thaliana</i>, GIGANTEA and the LOV domain of FKF1, to control synthetic zinc
finger transcription factor activity in human cells. Activation of
gene expression in human cells engineered with LITEZ was reversible
and repeatable by modulating the duration of illumination. The level
of gene expression could also be controlled by modulating light intensity.
Finally, gene expression could be activated in a spatially defined
pattern by illuminating the human cell culture through a photomask
of arbitrary geometry. LITEZ enables new approaches for precisely
regulating gene expression in biotechnology and medicine, as well
as studying gene function, cell–cell interactions, and tissue
morphogenesis
An Engineered Optogenetic Switch for Spatiotemporal Control of Gene Expression, Cell Differentiation, and Tissue Morphogenesis
The precise spatial and temporal
control of gene expression, cell differentiation, and tissue morphogenesis
has widespread application in regenerative medicine and the study
of tissue development. In this work, we applied optogenetics to control
cell differentiation and new tissue formation. Specifically, we engineered
an optogenetic “on” switch that provides permanent transgene
expression following a transient dose of blue light illumination.
To demonstrate its utility in controlling cell differentiation and
reprogramming, we incorporated an engineered form of the master myogenic
factor MyoD into this system in multipotent cells. Illumination of
cells with blue light activated myogenic differentiation, including
upregulation of myogenic markers and fusion into multinucleated myotubes.
Cell differentiation was spatially patterned by illumination of cell
cultures through a photomask. To demonstrate the application of the
system to controlling <i>in vivo</i> tissue development,
the light inducible switch was used to control the expression of VEGF
and angiopoietin-1, which induced angiogenic sprouting in a mouse
dorsal window chamber model. Live intravital microscopy showed illumination-dependent
increases in blood-perfused microvasculature. This optogenetic switch
is broadly useful for applications in which sustained and patterned
gene expression is desired following transient induction, including
tissue engineering, gene therapy, synthetic biology, and fundamental
studies of morphogenesis