Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay.
Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.
Center for Advanced Biotechnology and Medicine, Rutgers University.
Myka R
MR
Ababon
Department of Neuroscience and Cell Biology, Rutgers University.
Bo I
BI
Li
Center for Advanced Biotechnology and Medicine, Rutgers University.
Paul G
PG
Matteson
Center for Advanced Biotechnology and Medicine, Rutgers University; Department of Neuroscience and Cell Biology, Rutgers University; millonig@cabm-new.rutgers.edu.
James H
JH
Millonig
D000818:Animals; D002460:Cell Line; D004622:Embryo, Mammalian; D005260:Female; D017930:Genes, Reporter; D007763:Lac Operon; D008822:Mice, Transgenic; D011247:Pregnancy; D014212:Tretinoin; D001616:beta-Galactosidase
D014212:Tretinoin; D001616:beta-Galactosidase
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