NDLI: Imaging Tetrahymena ribozyme splicing activity in single live mammalian cells.

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Imaging Tetrahymena ribozyme splicing activity in single live mammalian cells.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Rao, Jianghong Hasegawa, Sumitaka Tsien, Roger Y. Jackson, W. Coyt
Description	Author Affiliation: Hasegawa S ( Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, University of California, Los Angeles, CA 90095-1770, USA.);
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.
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	25
Volume Number	100
Language	English
Publisher	National Academy of Sciences
Publisher Date	2003-12-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	RNA Splicing RNA, Catalytic Chemistry Tetrahymena Thermophila Metabolism Animals COS Cells Cell Separation Flow Cytometry Genes, Reporter Microscopy, Fluorescence Models, Chemical Molecular Sequence Data Nucleic Acid Conformation Open Reading Frames Plasmids Protein Structure, Tertiary RNA, Messenger Reverse Transcriptase Polymerase Chain Reaction Transfection Beta-Lactamases Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Multidisciplinary
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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