Photoactivated gene expression for cell fate mapping and cell manipulation.
A long-standing goal of developmental biologists is to create developmental fate maps by tracking individual cells through development. Another objective is to perturb the behavior of selected cells and follow the ensuing effects. To this end, we have developed a technique that allows for spatial and temporal control of gene expression in single cells or patches of cells using light to induce gene expression. This technique relies on "caging" the activity of the potent transcriptional activator GAL4VP16 with a photolabile compound, which can be removed with a brief exposure to long-wavelength ultraviolet (UV) light. The caged GAL4VP16 is injected into early-stage embryos, which are aged to the desired point in development, and the cell(s) of interest are irradiated with a brief pulse of long-wavelength UV light. This method has been used extensively in Drosophila, Xenopus, and Zebrafish embryos. The methods for purifying, caging, injection, and photoactivation of the GAL4VP16 protein, and methods for the visualization of marked cells are described in detail.
Minden
J
J
Department of Biological Sciences and Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, PA, USA. minden@cmu.edu
Namba
R
R
Mergliano
J
J
Cambridge
S
S
D000818:Animals; D019070:Cell Lineage / Q000235:genetics / Q000528:radiation effects*; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics / Q000528:radiation effects; D004625:Embryo, Nonmammalian / Q000737:chemistry / Q000166:cytology; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000528:radiation effects*; D017344:Genes, Insect / Q000235:genetics / Q000528:radiation effects; D010777:Photochemistry; D014982:Xenopus laevis / Q000196:embryology / Q000235:genetics; D015027:Zebrafish / Q000196:embryology / Q000235:genetics