Imaging Tetrahymena ribozyme splicing activity in single live mammalian cells. Export

@article{hasegawa03, 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.}, address = {Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, University of California, Los Angeles, CA 90095-1770, USA.}, author = {Hasegawa, S. and Jackson, W. C. and Tsien, R. Y. and Rao, J.}, citeulike-article-id = {3046350}, citeulike-linkout-0 = {http://dx.doi.org/10.1073/pnas.2036553100}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/14645710}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=14645710}, doi = {10.1073/pnas.2036553100}, issn = {0027-8424}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, keywords = {ribozyme, selection, tetrahymena}, month = {December}, number = {25}, pages = {14892--14896}, posted-at = {2008-07-27 18:31:18}, priority = {2}, title = {Imaging Tetrahymena ribozyme splicing activity in single live mammalian cells.}, url = {http://dx.doi.org/10.1073/pnas.2036553100}, volume = {100}, year = {2003} }

TY - JOUR ID - hasegawa03 L3 - citeulike-article-id:3046350 N2 - 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. IS - 25 JF - Proceedings of the National Academy of Sciences of the United States of America SN - 0027-8424 AD - Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, University of California, Los Angeles, CA 90095-1770, USA. EP - 14896 TI - Imaging Tetrahymena ribozyme splicing activity in single live mammalian cells. VL - 100 SP - 14892 KW - ribozyme KW - selection KW - tetrahymena AU - Hasegawa, S AU - Jackson, WC AU - Tsien, RY AU - Rao, J PY - 2003/12/09/ UR - http://dx.doi.org/10.1073/pnas.2036553100 ER -