Generation of Transgenic Mice Overexpressing a Hybrid Enzyme Up‐regulating Biosynthesis of Vascular Protector Prostacyclin

Abstract

Prostacyclin (PGI2) is a potent vascular protector through its anti‐platelet aggregation and vasodilation properties. Non‐steroid anti‐inflammatory drugs (NSAIDs) by inhibiting PGI2 production promote heart diseases. Recently, we have successfully engineered a hybrid enzyme (COX‐1‐10aa‐PGIS, by linking cyclooxygenase isoform 1(COX‐1) with PGI2 synthase (PGIS) through a 10 amino acids (10aa) linker) with triple catalytic functions directly converting arachidonic acid into PGI2. PGI2 biosynthesis was upregulated in the mammalian cells, transfected with the hybrid enzyme cDNA. Thus, with the objective of translating these in vitro results into in vivo, we generated transgenic mice overexpressing COX‐1‐10aa‐PGIS. PCR using mouse tail tissue confirmed the incorporation of COX‐1‐10aa‐PGIS gene in the transgenic mouse chromosome. Permanent expression of the hybrid enzyme was identified by Western blot. The catalytic activity of the hybrid enzyme to form PGI2 was determined by enzyme activity assay and LC/MS analysis of transgenic mouse tail and urine, respectively. In conclusion, a transgenic mouse overexpressing the hybrid enzyme with upregulated PGI2 biosynthesis was successfully obtained. The mice can be used to further uncover the mechanisms of the vascular protection of PGI2 and test the side effects of NSAIDs.Funding: R01 HL56712 (for KHR); RO1HL079389 (for KHR) and RC1 HL100807 (for RD and KHR)