Cloning, characterization and mapping of porcine CD14 reveals a high conservation of mammalian CD14 structure, expression and locus organization.

The cell surface protein CD14 plays a central role in innate immunity as a pattern recognition receptor. CD14 is part of a receptor complex also including toll-like receptor 4 and MD2 proteins. Binding of the ligand lipopolysaccharide to the complex on myeloid cells leads to release of pro-inflammatory cytokines and mediators from the cell. In this study, we present the cloning, characterization and tissue expression pattern of a porcine CD14 encoding cDNA, and the chromosomal localization of the porcine CD14 gene. The open reading frame is predicted to encode a protein of 373 amino acids, which shows conservation of structural as well as functional regions when compared to other mammalian species. The CD14 gene was localized to porcine chromosome 2 in a region syntenic to human chromosome 5q. Transcription analysis shows that CD14 is widely expressed in tissues examined in this study, which correlates well with expression primarily on myeloid cells.

Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.).

The expression levels of three commonly used housekeeping genes, EF1-alpha, RPS20 and Beta-Actin, were examined in seven different tissues and leucocytes from non-stimulated Atlantic salmon (Salmo salar L.). The tissues analysed by quantitative real-time PCR were gill, liver, intestine, muscle, spleen, head kidney leucocytes (HKL) and peripheral blood leucocytes (PBL). The experiments were performed to investigate the transcriptional stability within and between tissues and leucocytes and between individuals. For all tissues and leucocytes, an appropriate reference gene was identified except for muscle tissue. HKL were used as a calibrator and the expression of EF1-alpha varied maximally 2.5-fold in five out of the six tissues and leucocytes investigated relative to the expression of 18S rRNA. The RPS20 gene was more intermediate and varied at least by a factor of two and maximally by a 20-fold factor. Beta-Actin was generally the most regulated gene showing high variations for gill (5.8x) and spleen tissue (10.3x) relative to the calibrator. A suitable reference gene for muscle tissue was not found since the expression varied between 8.3- and 25-fold for the three genes compared to the calibrator. By comparing the expression results of the non-stimulated tissues and leucocytes using the Normfinder programme, it was further shown that EF1-alpha was the most stably expressed gene both between individuals and the different tissues/leucocytes. Stimulation with lipopolysaccharide (LPS) of TO cells and HKL from Atlantic salmon was additionally performed to reveal whether an immune stimulating agent would change the expression level of EF1-alpha, RPS20 and Beta-Actin. LPS stimulation of cells revealed that RPS20 and EF1-alpha were least regulated by the LPS treatment in the TO cells relative to 18S rRNA, but in HKL, Beta-Actin was the most appropriate gene. However, the variations were overall maximally two-fold in LPS-stimulated TO cells and HKL, which make all three genes suitable as reference genes in this case. A further experiment showed that no RT- and/or PCR inhibitors were present in the non-stimulated tissues and cells, indicating true transcriptional differences.

Mosaicism of the CAG repeat sequence in the Huntington disease gene in a pair of monozygotic twins.

We report on a pair of monozygotic twins belonging to a family segregating Huntington disease (HD). In routine DNA analysis of blood cells, they displayed three alleles of the CAG repeat sequence in the HD gene. Two different cell lines, carrying the normal allele together with either an expanded allele with 47 CAGs or an intermediate allele with 37 CAGs, were detected in blood and buccal epithelium from both twins. To our knowledge, this is the first case described of HD gene CAG repeat length mosaicism in blood cells. Haplotype analysis established that the 37 CAG allele most likely arose by contraction of the maternal 47 CAG allele. The contraction must have taken place postzygotically, possibly at a very early stage of development, and probably before separation of the twins. One of the twins has presented symptoms of HD for 4 years; his skin fibroblasts and hair roots carried only the cell line with the 47 CAG repeat allele. The other twin, who is without symptoms at present, displayed mosaicism in skin fibroblasts and hair roots. If the proportion of the two cell lines in the brain of each twin resembles that of their hair roots (another tissue originating from the ectoderm), the mosaicism in the unaffected twin would mean that only a part of his brain cells carried the expanded allele, which could explain why he, in contrast to his brother, has no symptoms at this time.