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  • Title: Design and expression of chimeric U1/ribozyme transgenes.
    Author: Abounader R, Montgomery R, Dietz H, Laterra J.
    Journal: Methods Mol Biol; 2004; 252():209-19. PubMed ID: 15017051.
    Abstract:
    The U1snRNA/ribozyme/antisense construct (designated U1/ribozyme) is a chimeric transgene that has proven to be very useful for inhibiting the expression of targeted genes in vitro and in vivo. It consists of a combination of hammerhead ribozyme flanked by target-specific antisense sequences that are in turn flanked by the loops and promoter of U1 snRNA. To construct U1/ribozymes, antisense/ribozyme sequences are first designed corresponding to ribozyme-cleavage consensus "GUC" sequences that are present in the targeted mRNA. Antisense/ribozyme sequences are then inserted between the U1 snRNA loops, and the conceptual secondary structure of the encoded regulatory RNA is analyzed to ensure proper folding. Appropriate antisense/ribozymes are then synthesized as oligonucleotides, annealed, and ligated into the pU1 vector containing the U1 snRNA promoter and loops to yield the pU1/ribozyme expression vector. Constructs can then be transiently or stably expressed in vitro and in vivo to inhibit the expression of target genes. U1/ribozymes can also be expressed in viral vectors for more efficient transfection, or complexed to liposomes for systemic delivery.
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