Direct selection for ribozyme cleavage activity in cells.
RNA
; 15(11): 2035-45, 2009 Nov.
Article
em En
| MEDLINE
| ID: mdl-19776159
ABSTRACT
Selection may prove to be a powerful tool for the generation of functional RNAs for in vivo genetic regulation. However, traditional in vitro selection schemes do not mimic physiological conditions, and in vivo selection schemes frequently use small pool sizes. Here we describe a hybrid in vitro/in vivo selection scheme that overcomes both of these disadvantages. In this new method, PCR-amplified expression templates are transfected into mammalian cells, transcribed hammerhead RNAs self-cleave, and the extracted, functional hammerhead ribozyme species are specifically amplified for the next round of selection. Using this method we have selected a number of cis-cleaving hammerhead ribozyme variants that are functional in vivo and lead to the inhibition of gene expression. More importantly, these results have led us to develop a quantitative, kinetic model that can be used to assess the stringency of the hybrid selection scheme and to direct future experiments.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Catalítico
/
Técnicas de Transferência de Genes
/
Técnicas de Amplificação de Ácido Nucleico
Tipo de estudo:
Evaluation_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
RNA
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
Estados Unidos