Down syndrome, the phenotypic expression of human trisomy 21, is presumed to result from a 1.5-fold increase in the expression of the genes on human chromosome 21. As an approach to the development of an animal model for Down syndrome, several strains of transgenic mice that carry the human Cu/Zn-superoxide dismutase gene have been prepared. These animals express the transgene in a manner similar to that of humans, with 0.9- and 0.7-kilobase transcripts in a 1:4 ratio, and synthesize the human enzyme in an active form capable of forming human-mouse enzyme heterodimers. Cu/Zn-superoxide superoxide dismutase activity is increased from 1.6- to 6.0-fold in the brains of four transgenic strains and to an equal or lesser extent in several other tissues. These animals provide a unique system for studying the consequences of increased dosage of the Cu/Zn-superoxide dismutase gene in Down syndrome and the role of this enzyme in a variety of other pathological processes.

A recombinant vaccinia virus that directs the synthesis of bacteriophage T7 RNA polymerase provides the basis for the expression of genes that are regulated by T7 promoters in mammalian cells. The T7 transcripts, which account for as much as 30% of the total cytoplasmic RNA at 24 hr after infection, are largely uncapped. To improve the translatability of the uncapped RNA, the encephalomyocarditis virus (EMCV) untranslated region (UTR) was inserted between the T7 promoter and the chloramphenicol acetyltransferase (CAT) gene. Experiments with a reticulocyte extract demonstrated that the EMCV UTR conferred efficient and cap-independent translatability to CAT RNA synthesized in vitro by T7 RNA polymerase. In cells infected with recombinant vaccinia viruses containing the T7 promoter-regulated CAT gene, the EMCV UTR increased the amount of CAT RNA on polyribosomes. The polyribosome-derived CAT RNA, which contained the EMCV UTR, was translated in vitro in a cap-independent fashion as well. Use of the EMCV UTR significantly enhanced the vaccinia/T7 hybrid expression system as it resulted in a 4- to 7-fold increase in total CAT activity. A further approximately 2-fold improvement was achieved by incubating the cells in hypertonic medium, which favors the translation of uncapped picornavirus RNA over cellular mRNAs. With this newly modified expression system, CAT was the predominant protein synthesized by infected cells and within 24 hr accounted for greater than 10% of the total cell protein.

Recombinant retroviral vectors producing multi-cistronic mRNAs were constructed. Picornavirus putative internal ribosome entry sites (IRES) were used to confer cap-independent translation of an Internal cistron. Internal clstrons were engineered by ligatlon of various lengths of the IRES of encephalomyocardltis (EMC) virus or polio virus to the E. coli chloramphenicol acetyltransferase (CAT) gene. The IRES/CAT fusions were introduced into retroviral vectors 3′ to the translation stop codon of the neomycin phosphotrans-ferase (NEO) gene, and the molecular constructs transfected into retroviral vector packaging lines. Retroviral vector producer cells efficiently express the internal CAT gene product only when the full length IRES is used. Both the EMC/CAT and polio/CAT retroviral vectors produced high tfter vector supernatant capable of productive transduction of target cells. To test the generality of this gene transfer system, a retroviral vector containing an IRES fusion to the human adenosine deaminase (ADA) gene was constructed. Producer cell supernatant was used to transduce NIH/3T3 cells, and transduced cells were shown to express NEO, and ADA. Novel three-gene-containing retroviral vectors were constructed by introducing the EMC/ADA fusion into either an existing internal-promoter-containing vector, or a polio/CAT biclstronic vector. Producer cell clones of the three-gene vectors synthesize all three gene products, were of high titer, and could productively transduce NIH/3T3 cells. By utilizing cap-independent translation units, IRES vectors can produce polycistronic mRNAs which enhance the ability of retrovlral-mediated gene transfer to engineer cells to produce multiple foreign proteins.