Cytoplasmic delivery of ribozymes leads to efficient reduction in alpha-lactalbumin mRNA levels in C127I mouse cells.
Ribozymes targeted to five sites along the alpha-lactalbumin (alpha-lac) mRNA were delivered to the cytoplasm of mouse C127I mammary cells using the T7-vaccinia virus delivery system and the amount of alpha-lac mRNA was monitored 24-48 h post-transfection. Three target sites were selected in the alpha-lac coding region (nucleotides 15, 145 and 361) and two were located in the 3' non-coding region (nucleotides 442 and 694). Acting in trans and at a target:ribozyme ratio of 1:1000, ribozymes targeting sites 361 and 694 reduced alpha-lac mRNA by > 80%; another two ribozymes (targeting nucleotides 442 and 145) reduced mRNA levels by 80 and 60% respectively; the fifth ribozyme (targeting nucleotide 15, near the AUG) was largely ineffective. The kinetic activity (kcat) of each ribozyme in vitro was somewhat predictive of the activity of the two ribozymes that targeted nucleotides 361 and 694, but was not predictive of the in vivo activity of the other three ribozymes. Down-regulation of the intracellular levels of alpha-lac paralleled the ribozyme-dependent reduction achieved for mRNA. For site 442, the reduction in both mRNA and protein was attributed to the catalytic activity of the ribozyme rather than to the antisense effects of the flanking arms, because delivery of an engineered (catalytically-inactive) variant had no effect on mRNA levels and a minimal effect on the level of alpha-lac present in the cell.
L'Huillier
P J
PJ
Centre for Gene Technology, School of Biological Sciences, University of Auckland, New Zealand.
Davis
S R
SR
Bellamy
A R
AR
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D000882:Haplorhini; D007768:Lactalbumin / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics*; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics; D014764:Viral Proteins
D016337:RNA, Catalytic; D012333:RNA, Messenger; D014764:Viral Proteins; D007768:Lactalbumin; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases