Design and expression of chimeric U1/ribozyme transgenes.
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.
Abounader
Roger
R
Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Montgomery
Robert
R
Dietz
Harry
H
Laterra
John
J
D000818:Animals; D001483:Base Sequence; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D016372:RNA, Antisense / Q000235:genetics; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics; D015183:Restriction Mapping / Q000379:methods; D019076:Transgenes / Q000235:genetics*
D016372:RNA, Antisense; D016337:RNA, Catalytic; D012342:RNA, Small Nuclear; C427204:hammerhead ribozyme