• Characterization of the mouse thromboxane synthase gene

      Zhang, Liqun; Shen, Rong-Fong (1997)
      Thromboxane synthase (TS) catalyzes the conversion of prostaglandin H2 (PGH2) to thromboxane A2 (TxA2), a potent inducer of platelet aggregation and vasoconstriction. Like many other prostaglandins, TxA2 exerts its biological activities in both an autocrine and paracrine fashion. In concert with prostacyclin, which is also derived from PGH2 and counteracts TxA2 activity, TxA2 plays a crucial role in the maintenance of hemostasis. A deficiency of TS has been found to cause bleeding disorders, whereas overproduction of TxA2 has been implicated in pathophysiological conditions, such as cardiovascular diseases, asthma, renal diseases, and immune disorders. The in vivo functions of TxA2 are not presently well-defined due, in part, to the lack of an animal model defective in TS. The goal of this study was to generate a mouse model of TS deficiency through homologous recombination ("knockout") for better assessment of TS function in vivo. The cDNA for mouse TS was first isolated and characterized. It encodes a 58 kD protein sharing 80% identity with human TS. The genomic fragments encoding mouse TS gene were isolated and characterized, from bacteriophage lambda and P1 libraries, using mouse TS cDNA as the probe. The entire gene consists of 13 exons spanning greater than 150 kb DNA. Using fluorescence in situ hybridization, the gene was also mapped to mouse chromosome 6. Studies on the expression of the mouse TS gene indicated that it is transcribed from multiple transcription start sites. The level of TS gene expression is modulated by several upstream repressive elements and a short (70 bp) positive regulatory element containing an AP-1/NF-E2 binding site. Finally, to achieve the targeted disruption of mouse TS gene, a replacement construct containing both positive and negative selectable markers, neo and HSV-tk, respectively, was introduced into mouse embryonic stem (ES) cells, and the transfected cells selected by both G418 and gancyclovir. Two independent ES clones were found to be homologous recombinants, as evidenced by a defective TS allele revealed through Southern analysis. Microinjection of the ES cells into blastocysts has led to the live birth of several chimeric mice. Collectively, our studies established the genetic basis for TS gene research in mice, and will expedite the understanding of the contribution of TS in normal physiology as well as in disease states.