Abstract |
The U6 expression system was explored for efficient expression of a ribozyme against the human proto-oncogene c-neu. A hammerhead ribozyme (neuRz) and the control mutant ribozyme (MRz) were targeted to cleave c-neu mRNA at the tyrosine kinase domain. In vitro cleavage showed that neuRz was very active while MRz was not. Near-maximal target cleavage observed at a low ribozyme:target ratio (0.1) suggests that neuRz has good activity and turnover capability under physiological conditions, i.e., <5 mM MgCl(2) and 37 degrees C. Chimeric U6 ribozyme was expressed at about 5 x 10(6) copies/cell at 48 h in the ovarian carcinoma cell line SKOV-3.ip1. Partial down-regulation of c-neu mRNA and protein was observed in a dose-dependent manner in cells transiently transfected with U6neuRz- but not with MRz-containing plasmid. Sorted transient transfectants demonstrated dramatic growth inhibition with the neuRz-expressing cells. Our results demonstrate that the U6 expression system is very efficient and suitable for the expression of a hammerhead ribozyme. Moreover, nonviral delivery of the neuRz-expressing plasmid resulted in specific down-regulation of c-neu and, subsequently, growth inhibition of ovarian cancer cells overexpressing c-neu.
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Authors | V W Lui, Y He, L Huang |
Journal | Molecular therapy : the journal of the American Society of Gene Therapy
(Mol Ther)
Vol. 3
Issue 2
Pg. 169-77
(Feb 2001)
ISSN: 1525-0016 [Print] United States |
PMID | 11237673
(Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
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Chemical References |
- MAS1 protein, human
- Proto-Oncogene Mas
- RNA, Messenger
- Ribonucleoprotein, U4-U6 Small Nuclear
- Protein-Tyrosine Kinases
- Receptor, ErbB-2
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Topics |
- Blotting, Northern
- Cell Division
- Cell Separation
- Dose-Response Relationship, Drug
- Down-Regulation
- Female
- Flow Cytometry
- Genetic Therapy
(methods)
- Humans
- Ovarian Neoplasms
(metabolism)
- Plasmids
(metabolism)
- Promoter Regions, Genetic
- Protein Structure, Tertiary
- Protein-Tyrosine Kinases
(chemistry)
- Proto-Oncogene Mas
- RNA, Messenger
(metabolism)
- Receptor, ErbB-2
(metabolism)
- Ribonucleoprotein, U4-U6 Small Nuclear
(metabolism)
- Time Factors
- Transfection
- Tumor Cells, Cultured
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