Genzyme Corporation

Tsi, Inc.

Exemplar Corporation

Cha-Mer Wei

Vermuri B Reddy

Nancy Hsiung

William R Dackowski

Jeffrey F Lemontt

Edward S Cole

Richard Douglas

David Tai-Yui Lau

Richard D Purcell, Jr

A rapid, sensitive, and quantitative method and assay system using transgenic animal cells and adult or embryonic transgenic animals to rapidly screen compounds for mutagenic or teratogenic activity. Each cell from a cell culture, a differentiated tissue from an animal or an animal embryo contains a target gene and a reporter gene, both including an animal promoter/enhancer, a coding sequence and a transcription termination signal, so that they are capable of expression in animal cells. The product of the target gene is capable of regulating expression of the reporter gene through interaction with a regulatory sequence in the reporter gene. In the preferred embodiment, the target gene lacI (lac repressor) is coupled to the reporter gene lacZ encoding beta-galactosidase, which is easily detectable by cytochemical or histochemical procedures. When the animal cell is exposed to a compound causing a mutation either altering expression of the target gene or altering the operator of the reporter gene, the reporter gene is expressed.

Rapid screening mutagenesis and teratogenesis assay

Mutants of human urokinase are produced which have an altered amino acid sequence in the domain responsible for binding of plasminogen activator inhibitor(s). One example of an inhibitor resistant mutant is described in detail. Six amino acids (179-184), R H R G G S, have been deleted from the mature urokinase. The gene encoding inhibitor resistant preprourokinase is chemically synthesized according to computer-designed nucleotide sequences containing convenient restriction endonuclease cleavage sites, a signal for the initiation of translation, a sequence encoding the signal peptide of mouse whey acid protein and a complete coding sequence for mature inhibitor-resistant urokinase. The gene was used to transform cultured mouse cells to produce clones that stably incorporate the gene in the genome. Clones with high levels of expression were used as the hosts for production of this protein. Alternatively, the DNA for the inhibitor-resistant prourokinase/urokinase, in combination with a tissue specific promoter, could be introduced into fertilized embryos, the embryo implanted into a suitably prepared female of the same species, and the offspring analyzed for presence of the prourokinase/urokinase gene. The transgenic animals can then be bred and the urokinase produced in the milk.

Inhibitor-resistant urokinase

A replicable expression vector capable, in a transformed host mammallian cell, of expressing a DNA sequence encoding active human TPA which, when expressed in the host cell, yields active TPA which lacks one or more of the TPA-bound N-linked carbohydrate moieties of naturally occurring human TPA.

Human uterine tissue plasminogen activator produced by recombinant DNA

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Worcester, MA, United States of America

Cha-Mer Wei