REGULATION OF CAT PROTEIN BY RIBOZYME AND ANTISENSE MESSENGER-RNA IN TRANSGENIC MICE

Transgenic mouse lines were engineered to express stably antisense mRN A or antisense mRNA containing catalytic ribozyme (rbz) structures com plementary to bacterial chloramphenicol acetyltransferase (CAT) gene t ranscripts. One transgenic line expressed antisense mRNA that specific ally targeted full-length CAT coding sequences (ACAT). Another transge nic line expressed full-length antisense CAT mRNA which was modified b y mutagensis to include four rbz cassettes (rbz-ACAT) in order to comp are antisense versus antisense-rbz function in vivo. Preliminary data were also collected from a transgenic mouse line expressing antisense mRNA targeting 72% of the 5' region of CAT coding sequences (5' ACAT). All constructs contained similar control elements in their design. Pr omoter elements were derived from the bovine alpha(s1)-casein gene, wh ile the small t intron and 3' control sequences were derived from SV40 . The ability of these various constructs to down-regulate CAT protein levels was compared by analysis of CAT protein production in lactatin g double-hemizygous transgenic female mice. Every double-hemizygous mo use analysed expressed mRNA from the alpha(s1)-casein-CAT construct (C larke et al., 1994) and equivalent levels of mRNA from one of the thre e antisense constructs. Transgenic mouse lines expressing both ACAT an d CAT mRNA down-regulated CAT protein levels by 90% of that found in t he CAT only transgenic population. Similarly, double-hemizygous transg enic lines expressing both rbz-ACAT and CAT mRNA regulated CAT protein levels by 87%. Preliminary data suggests that expression of mRNA from 5' ACAT/CAT double-hemizygote mice allowed approximately 67% down-reg ulation of normal CAT protein levels. We conclude that incorporation o f multiple ribozymes within the full-length antisense CAT construct do es not enhance the effectiveness of antisense mRNA in the down-regulat ion of CAT protein production in our system.