A Novel Fluorescent-Based Screen to Identify Small Synthetic Internal Ribosome Entry Site (IRES) Elements
Two-color reporter system to rapidly and efficiently determine hits Small synthetic IRES elements are changeable in length and sequence to accommodate a random mutagenesis approach or a directed design approach Small IRES size (<60 nt) allows for the incorporation of larger (or multiple) genes into a single retroviral vector for gene therapeutics Utilization of protoplast fusion for plasmid delivery imparts greater efficiency and dramatically lowers background noise
For the use in IRES element detection Identify trans-acting translation factors, alter cellular metabolism and augment current gene therapy applications
Researchers at UCLA have created an assay to identify IRES elements from a library of bicistronic plasmids encoding two fluorescent proteins separated by a small (<60 nt) synthetic intervening sequence of changeable length. By using fluorescence-activated cell-sorting techniques, it is possible to determine candidate IRES element sequences for further analysis.
Patent Number: US6833254B2
Application Number: US200287171A
Inventor: Dasgupta, Asim | Venkatesan, Arun
Priority Date: 1 Mar 2001
Priority Number: US6833254B2
Application Date: 1 Mar 2002
Publication Date: 21 Dec 2004
IPC Current: C12N001510 | C12N001521
US Class: 4350691 | 435005 | 435039 | 4353201 | 435334 | 435006
Assignee Applicant: The Regents of the University of California
Title: Method to identify IRES elements
Usefulness: Method to identify IRES elements
Summary: The method is useful for identifying trans-acting translation factors or IRES elements, which are useful for controlling viral infection in a cell or regulating cellular metabolism.
Novelty: Identifying an internal ribosome entry site (IRES) element for controlling viral infection or regulating metabolism in cells by employing a bicistronic system for reporter proteins involving both cap- and IRES-mediated translation
Biomedical
DNA/Gene Engineering
6833254
State Of Development The screen has been designed, tested and validated and two candidates, with no sequence or structural homology to known viral and cellular IRES elements, were shown to specifically block poliovirus and hepatitis C virus IRES-mediated translation. Background Internal ribosome entry site (IRES)-mediated translation initiates and assembles translation machinery at a site close to the start codon in a manner that does not require the traditional eukaryotic 5 nucleotide cap or eIF4E (cap-binding protein). Initially discovered in picornaviruses (poliovirus, encephalomyocarditis virus, rhinovirus, etc.), viral IRES-mediated translation relies on virus-specific combinations of transcription factors, trans-acting factors that help stabilize the IRES structure and the IRES elements themselves, which can span hundreds of nucleotides in length and have complicated secondary and tertiary structures. The complex way in which the IRES elements mediate translation is of great importance to researchers seeking insight into viral gene expression and ultimately, anti-viral therapeutics. Related Materials Tech ID/UC Case 20057/2001-202-0 Related Cases 2001-202-0
USA
