In vitro cleavage of collagen mRNA in Ito cells by ribozyme

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1. Purification of lto cellsRat lto cells were separated by centrifugation using metrizamide solution after perfusion and digestion of the liver with collagenase.2. Synthesis of target RNARat type l collagen alpha (1)cDNA was inserted into pBluescript and sequenced by dye primer method. It was confirmed that this cDNA had Gly-X-Y sequence, specific for collagen genes. The target RNA was synthesized by in vitro transcriprtion using [^<32>P]-UTP.3. Synthesis of ribozymeThe several regions with GUC sequence in target RNA were selected and hammerhead ribozyme complimentary to such regions was designed. The ribozyme was synthesized by in vitro transcription from template DNA,which was made from PCR products using chemically synthesized oligonucleotides.4. In vitro cleavage of collagenmRNA by ribozymeThe target RNA (collagen mRNA) was incubated with ribozyme at 37ﾟC and the cleaved fragments of collagen mRNA were detected by electrophoresis and autoradiograph. The cleavage activity was detected since 30 min after incubation and approximately 70% of collagen mRNA was cleaved at maximum. The optimal conditions were a target/ribozyme ratio of 1/1 (molar basis) and a MgCl_2 concentration of 10 mM.5. Effect of ribozyme on expression of collagen mRNA in lto cellsIto cells were incubated with the addition of ribozyme and the expression of collagen mRNA was determined by Northern blotting using radiolabeled oligo DNA prove complimentary to collagen mRNA.The cleavage activity was not sufficiently detected. We are planning the transfer of ribozyme into lto cells using adenovirus vectors.