Generation of CYP4A1 transgenic rats using the Sleeping Beauty transposon system

Abstract

Cytochrome P450 enzymes of the 4A family that catalyze the formation of 20-HETE have been implicated in the regulation of renal function, vascular tone on the long term control of blood pressure. To better determine the role of CYP4A1 in the development of hypertension and renal and vascular dysfunction, we utilized the Sleeping Beauty (SB) transposon system to generate CYP4A1 transgenic rats. Rat full length CYP4A1 cDNA was inserted into pT2 transposon plasmid containing a hybrid chicken β-actin promoter fused with elements of the CMV enhancer (CAG). Pronuclei of the fertilizedeggs collected from Dahl SS female rats are microinjectedwith a circular SB transposon-plasmid construct along with transposase capped RNA transcripts and incubated overnight. Then 2-cell staged oocytes were transferred intothe oviducts of pseudopregnant foster Dahl females. Transgenic founders were identified by PCR using both CYP4A1 and transgene-specific primers. The insertion sites were checked by Ligation-mediated PCR and 5′-rapid amplification of cDNA ends (5′RACE). Heterozygous founders were backcrossed to Dahl SS rats and the progeny were brother sister mated to derive two homozygous-transgenic lines. The production of 20-HETE in the renal cortex is elevated by 3 fold in the CYP4A1 transgenic line in comparison to SS rats. These animals will be in exploring the role of CYP4A1 in the pathogenesis of hypertension and renal and cardiovascular end organ damage. HL36279 and HL29587