Inhibition of kit expression in P815 mouse mastocytoma cells by a hammerhead ribozyme.

Abstract

Using P815 cells, we designed and tested an antisense hammerhead ribozyme for its ability to mediate cleavage of mouse c-kit mRNA at the transmembrane-encoding region. The ribozyme demonstrated in vitro cis cleavage of extraneous 5' sequence in the ribozyme construct as well as trans cleavage of a target c-kit mRNA sequence. The in vitro cleavage was effective at 37 degrees C. A subpopulation of P815 cells transfected with the pCDNA1 plasmid-containing the anti-c-kit ribozyme demonstrated inhibited Kit expression as indicated by flow cytometry, but the inhibition could not be maintained over time. The anti-c-kit ribozyme was put under the control of a tetracycline-inducible two-plasmid system of expression. Incomplete inhibition of Kit expression was observed when transcription of the ribozyme was allowed by the absence of tetracycline, but not when tetracycline was present. Finally, complete inhibition of Kit expression was observed when pCDNA1-ribozyme-transfected cells were analyzed immediately after enrichment by cotransfection with a plasmid expressing green fluorescent protein, followed by cell sorting. The results suggest that anti-c-kit ribozymes might be useful for inhibiting Kit expression in hyperplastic mast cells, if delivery of ribozymes can be optimized.