Regulation of α1,3galactosyltransferase expression in pig endothelial cells

Abstract

The disaccharide galactosea1,3galactose (the aGal epitope) is the major xenoantigen responsible for the hyperacute vascular rejection occurring in pig-to-primates organ transplantation. The synthesis of the aGal epitope is catalyzed by the enzyme a1,3-galactosyltransferase (a1,3GalT). To be able to control porcine a1,3GalT gene expression specifically, we have analyzed the upstream portion of the a1,3GalT gene, and identified the regulatory sequences.Porcine a1,3GalT transcripts were detected by 5¢ RACE analysis, and the corresponding genomic sequences were isolated from a phage library. The porcine a1,3GalT gene consists of at least 10 different exons, four of which contain 5¢ untranslated sequence. Four distinct promoters, termed A-D, drive a1,3GalT gene transcription in porcine cells. A combination of alternative promoter usage and alternative splicing produces a series of transcripts that differ in their 5¢ portion, but encode the same protein.Promoters A-C have been isolated, and functionally characterized using luciferase reporter gene assays in transfected porcine endothelial cells (PEC-A). Promoter preference in porcine endothelial cells was estimated on the basis of relative transcript levels as determined by real-time quantitative PCR. More than 90% of the a1,3GalT transcripts in PEC-A cells originate from promoter B, which has characteristics of a housekeeping gene promoter. While promoter preference remains unchanged, a1,3GalT mRNA levels increase by 50% in 12 h upon tumour necrosis factor a-activation of PEC-A cells. However, the magnitude of this change induced by inflammatory conditions could be insufficient to affect cell surface a1,3-galactosylation.

Advances in Swine Transcriptomics

“…The QPCR technique can, of course, be used to measure gene expression independently of microarray confirmation. Examples where 4 or more genes were quantitatively assayed with or without microarray data include measurement of response to LPS 36 , to infection 26 , 29 , 37 , 38 , or to islet graft rejection 39 ; measurement of promoter activity in vitro 40 , 41 ; assessing expression of gene family transcript isoforms 42 ; measuring responses during parthenogenesis 43 , 44 or oocyte/conceptus development 23 , 45 ; brain response to early weaning/isolation 46 , 47 ; multi-tissue response to carnitine treatment 48 or liver response to dietary treatments 28 , 49 ; differences across specific muscle types 50 ; and differences between stented and unstented arteries 51 .…”

Approach for defining endogenous reference genes in gene expression experiments

“…However, rRNAs can be used only when total RNA is isolated and the cDNA synthesis is primed with random hexamers. In some studies plasmids containing the amplicon of the target or endogenous reference gene have been used to generate a standard curve and express the data as a relative quantiWcation of absolute data [3,4].…”