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Imaging Tetrahymena ribozyme splicing activity in single live mammalian cells.

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.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Hasegawa S,Jackson WC,Tsien RY,Rao J

doi

10.1073/pnas.2036553100

keywords:

subject

Has Abstract

pub_date

2003-12-09 00:00:00

pages

14892-6

issue

25

eissn

0027-8424

issn

1091-6490

pii

2036553100

journal_volume

100

pub_type

杂志文章

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