Ribozymes are small RNA molecules that cleave specific RNA molecules enzymatically. Ribozymes can be designed to target specific RNA molecules, thereby modulating gene expression. A hammerhead ribozyme was designed to cleave inhibin $\alpha$ subunit mRNA. This ribozyme was synthesized as double-stranded oligodeoxynucleotides and cloned into plasmid pGEM-4Z. In vitro transcripts of this cloned ribozyme and a cloned rat inhibin $\alpha$ subunit cDNA were incubated together both at 50$\sp\circ$C and at 37$\sp\circ$C. Northern blot analysis revealed two extra bands of the expected size representing the cleavage products. The cleavage was specific. No cleavage was observed when in vitro antisense transcripts of rat inhibin $\alpha$ subunit cDNA were incubated with the ribozyme. Since our goal is to down-regulate inhibin production using ribozyme, a DNA construct carrying the ribozyme was constructed to generate transgenic mice. The ribozyme was connected to the 3$\sp\prime$ end of the E. coli lacZ gene, and was placed under the control of the H-2K$\sp{\rm b}$ gene promoter. Two transgenic mouse lines were established from one transgenic founder, because the injected DNA integrated at two sites, which segregated independently from each other. In one line lacZ expression was detected in tissues including the cornea, the snout, the tongue, the palm of paws, the ventral midline, the pituitary, the dorsum near the spinal cord, on day 13 embryos. Northern blot analysis indicated that ribozyme expression was detected in adult spleen. These results demonstrated that (1) this engineered ribozyme cleaved inhibin $\alpha$ subunit transcripts in vitro, suggesting that there were no secondary structures which interfere with the cleavage activity of the ribozyme; (2) two transgenic mouse lines carrying the ribozyme were established, and expression of the ribozyme was determined.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8724 |
| Date | 01 January 1993 |
| Creators | Ge, Lisheng |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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