Abstracts / Prostaglandins & other Lipid Mediators 59 (1999) 1-235 5

ENDOTOXIN INDUCTION OF LUCIFERASE ACTIVITY IN MICE TRANSGENIC FOR A COX-2 PROMOTER-LUCIFERASE REPORTER CONSTRUCT

Amanda Freeman’, Harvey Herschmar?, Olga Voznesenskyl, Anita Bhatt’, Steve Clark’, and Carol Pilbeam’

‘Univ. of Conn. Health Ctr., Farmington, CT 06030, USA, and 2University of Califomia School of Medicine, Los Angeles, CA 90024,USA

Induction of COX-2 expression by many agonists is largely transcriptional and mediated by elements within the -371 bp of the 5’-flanking region proximal to the transcription start site. We developed 4 lines of mice, via pronuclear microinjection, transgenic for -37 1/+70 bp of the S- flanking DNA of the murine COX-2 gene fused to a luciferase reporter gene (Pluc371). To examine Pluc regulation in vivo, mice were injected IP with vehicle ór lipopolysaccharide (LPS; 4 mg). Mice were sacrificed 3.5 h after injection, and tissues extracted for luciferase activity and total RNA. Luciferase activity was measured in soluble tissue extracts, normalized to total protein, and expressed as Relative Light Units (RLU)/hg protein/s. Constitutive Pluc activity varied greatly among different tissues, but activity for a particular tissue was reproduc- ible among animals. Constitutive activity, in RLU/p.g protein/s, was 200-500 in bram; 20-50 in calvariae and uterus/ovary; 3-8 in lung, spleen and heart; and cl.0 in kidney and smal1 bowel. After LPS injection, Pluc 1 activity was increased 15-fold in calvariae; 13-fold in uterus/ovary; lO- to 12-fold in heart and smal1 bowel; 4- to 9-fold in kidney, lung and spleen; and only 1.5-fold in brain. Similar results were obtained in two different lines of Pluc 1 mice. Hence, the highest constitutive Pluc 1 activity and the smallest LPS-induced increase in vivo were in bram; while the largest fold-induction after LPS injection was in calvarial bone. Luciferase activities wil1 be correlated with Northem analyses of luciferase and endogenous COX-2 mRNA expression.

[Supported by NIH grants DK48361 to CP and AI34567 to HH.]