Characterization of porcine nuclear receptors and their splice variants.

摘要

This thesis is the investigation of porcine orthologs of common nuclear receptors and their splice variants. It was found using a dualluciferase reporter assay, porcine PXR (pgPXR-WT) and human PXR (hPXR) responded to 12 common ligands, with similar levels of activation occurring for 6 of these. It was found that pgFXR-WT significantly responded to 3 ligands, two of which also significantly activated hFXR, although the degree of response was not significantly similar between species. 3-methylindole (skatole) was identified as a novel inverse agonist for pgPXR-WT, pgFXR-WT, and porcine constitutive androstane receptor (pgCAR). Through the course of cloning pgFXR, 5 novel splice variants were isolated, while splice variants of pgPXR and pgCAR had been isolated previously (Pollock et al., 2007 Gray et al., 2009). It was found that the pgPXR variants were present in liver cDNA samples from 3.33% to 7.92% of the total pgPXR, while the pgFXR variants were present from 1.92% to 9.26% of total pgFXR. The pgCAR variants were found to comprise of from 4.61% to 9.20% of the total pgCAR. In the presence of the wild-type protein it was found that pgCAR-SV2 exerted a significant and dose dependent dominant negative effect on wild-type pgCAR. pgPXR-SVI had a significant and dose dependent dominant positive effect on wild-type pgPXR, while pgFXR-SVI had a significant and dose dependent dominant positive effect on wild-type pgFXR. The characterization of these porcine nuclear receptors is important to determine how they differ from other animal systems, so specific targeting in pigs can be carried out.