Use of an antisense oligonucleotide to inhibit expression of a mutated human procollagen gene (COL1A1) in transfected mouse 3T3 cells.
A series of antisense oligonucleotides were developed to inhibit specifically expression of a mutated exogenous gene for collagen without inhibiting expression of an endogenous gene for the same protein. The test system consisted of mouse NIH 3T3 cells that were stably transfected with an internally deleted construct of the human gene for the pro alpha 1(I) chain of type I procollagen [Olsen et al. (1991) J. Biol. Chem. 266, 1117]. The target site was a region at the 3' end of exon 1 and the first few nucleotides of intron 1 of the exogenous human gene that differed in sequence by nine nucleotides from the sequence of the endogenous mouse gene. Expression of the two genes was assayed by Western blot with cross-reacting antibodies and by steady-state levels of mRNAs. None of the oligonucleotides were effective in concentrations up to 25 microM when administered without any carrier. However, when administered with 5 or 10 micrograms/mL lipofectin, one of the oligonucleotides in concentrations of 0.1-0.2 microM inhibited expression of the exogenous gene from 50% to 80% without significant inhibition of expression of the endogenous gene. Also, a missense version of the same oligonucleotide had no significant effect, and the inhibition observed with the most effective oligonucleotide was abolished by a single base change. Time course experiments indicated that, after a 4-h treatment, inhibition appeared at 8 h and persisted for at least 22 h.(ABSTRACT TRUNCATED AT 250 WORDS)
Colige
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A
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Philadelphia, Pennsylvania 19107.
Sokolov
B P
BP
Nugent
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P
Baserga
R
R
Prockop
D J
DJ
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D005091:Exons; D005353:Fibronectins / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007438:Introns; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011347:Procollagen / Q000235:genetics*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D014162:Transfection
D005353:Fibronectins; D016376:Oligonucleotides, Antisense; D010714:Phosphatidylethanolamines; D011347:Procollagen; D012333:RNA, Messenger; C020888:1,2-dielaidoylphosphatidylethanolamine