1. Genetically encoded betaxanthin-based small-molecular fluorescent reporter for mammalian cells
- Author
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Martin Fussenegger, David W Fuchs, Tobias Strittmatter, Pascal Stücheli, Pratik Saxena, Simon Sieber, Leo Scheller, Karl Gademann, University of Zurich, and Fussenegger, Martin
- Subjects
10120 Department of Chemistry ,AcademicSubjects/SCI00010 ,dopa-dioxygenase ,01 natural sciences ,stress ,Narese/2 ,Genes, Reporter ,Narese/1 ,Cricetinae ,540 Chemistry ,Gene expression ,Picolinic Acids ,time ,chemistry.chemical_classification ,0303 health sciences ,Chinese hamster ovary cell ,Absorption, Radiation ,Recombinant Proteins ,Biochemistry ,Oxygenases ,Methods Online ,Single-Cell Analysis ,Tyrosine 3-Monooxygenase ,Ultraviolet Rays ,proliferation ,pigments ,Heterologous ,CHO Cells ,Biology ,Fungal Proteins ,03 medical and health sciences ,Cricetulus ,1311 Genetics ,Genetics ,Animals ,Humans ,Viability assay ,030304 developmental biology ,010405 organic chemistry ,HEK 293 cells ,gene-expression ,proteins ,0104 chemical sciences ,HEK293 Cells ,Spectrometry, Fluorescence ,chemistry ,Microscopy, Fluorescence ,Cell culture ,betalains ,biosynthesis ,mCherry ,Glycoprotein - Abstract
We designed and engineered a dye production cassette encoding a heterologous pathway, including human tyrosine hydroxylase and Amanita muscaria 4,5-DOPA dioxygenase, for the biosynthesis of the betaxanthin family of plant and fungal pigments in mammalian cells. The system does not impair cell viability, and can be used as a non-protein reporter system to directly visualize the dynamics of gene expression by profiling absorbance or fluorescence in the supernatant of cell cultures, as well as for fluorescence labeling of individual cells. Pigment profiling can also be multiplexed with reporter proteins such as mCherry or the human model glycoprotein SEAP (secreted alkaline phosphatase). Furthermore, absorbance measurement with a smartphone camera using standard application software enables inexpensive, low-tech reporter quantification., Nucleic Acids Research, 48 (12), ISSN:1362-4962, ISSN:0301-5610
- Published
- 2020