Imaging Tetrahymena ribozyme splicing activity in single live mammalian cells
AUTOR(ES)
Hasegawa, Sumitaka
FONTE
National Academy of Sciences
RESUMO
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, β-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 β-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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=299846Documentos Relacionados
- A Tetrahymena thermophila ribozyme-based indicator gene to detect transposition of marked retroelements in mammalian cells
- Quantitative Imaging of Single Live Cells Reveals Spatiotemporal Dynamics of Multistep Signaling Events of Chemoattractant Gradient Sensing in DictyosteliumD⃞V⃞
- Experimental evidence for RNA trans-splicing in mammalian cells.
- Digital imaging of free calcium changes and of spatial gradients in growing processes in single, mammalian central nervous system cells.
- P2 functions as a spacer in the Tetrahymena ribozyme.