Identification of eukaryotic internal ribosome entry site (IRES) elements
US8257945B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Sep 3, 2002 |
| Grant date | Sep 4, 2012 |
| Priority date | — |
| Expiry date | May 17, 2026 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12N2510/00
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
A method of search for and identification of a eukaryotic IRES element active in cap-independent translation of RNA in eukaryotic cells is provided, comprising the following steps: i) screening eukaryotic mRNA sequences or corresponding DNA sequences for a potential IRES element having a block of nucleotides having: a) a length of at least 30 nucleotides; b) an adenine nucleotide content of at least 40 mol-%; and c) a pyrimidine nucleotide content of less than 40 mol-%; ii) inserting said potential IRES element into a linear dicistronic construct between an upstream gene and a downstream GUS reporter gene, whereby said potential IRES element is positioned for IRES-dependent translation of said downstream GUS gene and whereby said upstream gene is preceded by a stable hairpin structure to prevent IRES-independent translation of said genes; and iii) testing said potential IRES element for IRES-dependent translation of said GUS gene in a rabbit reticulocyte lysate or in a wheat germ extract in vitro translation assay, whereby GUS gene expression is quantitated preferably relative to a construct having a reference IRES element or a non-IRES element between said upstream gene and said G…
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