ArticleProceedings of the National Academy of Sciences of the United States of America2003
Imaging Tetrahymena ribozyme splicing activity in single live mammalian cells.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2003. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
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Who cites it
6 citing papers in PubMed, 38 citations in OpenAlex.
- Article
- Determination of melatonin by a whole cell bioassay in fermented beverages.Scientific reports · 2019Article
- Imaging of pre-mRNA splicing in living subjects using a genetically encoded luciferase reporter.Biomedical optics express · 2018Article
- Evaluating target silencing by short hairpin RNA mediated by the group I intron in cultured mammalian cells.BMC biotechnology · 2011Article
- A medaka model of cancer allowing direct observation of transplanted tumor cells in vivo at a cellular-level resolution.Proceedings of the National Academy of Sciences of the United States of America · 2009Article
- Flow cytometric detection of specific RNAs in native human cells with quenched autoligating FRET probes.Proceedings of the National Academy of Sciences of the United States of America · 2006Article
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Authors and funding
4 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Tetrahymena ribozymes hold promise for repairing genetic disorders but are largely limited by their modest splicing efficiency and low production of final therapeutic proteins. Ribozyme evolution in intact living mammalian cells would greatly facilitate the discovery of new ribozyme variants with high in vivo activity, but no such strategies have been reported. Here we present a study using a new reporter enzyme, beta-lactamase, to report splicing activity in single living cells and perform high-throughput screening with flow cytometry. The reporter ribozyme constructs consist of the self-splicing Tetrahymena thermophila group I intron ribozyme that is inserted into the ORF of the mRNA of beta-lactamase. The splicing activity in single living cells can be readily detected quantitatively and visualized. Individual cells have shown considerable heterogeneity in ribozyme activity. Screening of Tetrahymena ribozymes with insertions in the middle of the L1 loop led to identification of better variants with at least 4-fold more final in vivo activity than the native sequence. Our work has provided a new reporter system that allows high-throughput screening with flow cytometry of single living mammalian cells for a direct and facile in vivo selection of desired ribozyme variants.
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