ABSTRACT
The origins, design, fabrication and performance of an Atlantic salmon microarray are described. The microarray comprises 16 950 Atlantic salmon-derived cDNA features, printed in duplicate and mostly sourced from pre-existing expressed sequence tag (EST) collections [SALGENE and salmon genome project (SGP)] but also supplemented with cDNAs from suppression subtractive hybridization libraries and candidate genes involved in immune response, protein catabolism, lipid metabolism and the parr-smolt transformation. A preliminary analysis of a dietary lipid experiment identified a number of genes known to be involved in lipid metabolism. Significant fold change differences (as low as 1.2x) were apparent from the microarray analysis and were confirmed by quantitative real-time polymerase chain reaction analysis. The study also highlighted the potential for obtaining artefactual expression patterns as a result of cross-hybridization of similar transcripts. Examination of the robustness and sensitivity of the experimental design employed demonstrated the greater importance of biological replication over technical (dye flip) replication for identification of a limited number of key genes in the studied system. The TRAITS (TRanscriptome Analysis of Important Traits of Salmon)-salmon genome project microarray has been proven, in a number of studies, to be a powerful tool for the study of key traits of Atlantic salmon biology. It is now available for use by researchers in the wider scientific community.
ABSTRACT
Granulated metrial gland (GMG) cells are estrogen-receptor and Interleukin 2 (IL-2) receptor positive lymphocytes of the Natural Killer cell lineage found in the murine uterus during pregnancy. Functional studies of these cells, which are now more frequently called uterine NK (uNK) cells, have been limited due to technical difficulties. The cells are difficult to isolate and their proliferation and differentiation have not been achieved in culture. In 1988, Mukhtar and Stewart (Cell Tiss. Res., 253, 413-417) reported a method for explant culture of metrial glands isolated from pregnant rodents that yielded an almost pure population of uNK cells. This major technical advance has supported most of the subsequent functional and molecular studies of rodent uNK cells. However, the quality of the cells isolated by the explant culture procedure has not been established. A cytochemical approach was used to identify and quantify the cells migrating from metrial glands. At midpregnancy, almost all (> 90%) migrating nucleated cells were NK cells. Earlier in gestation, a significant proportion (25%) of cells having lymphoid morphology could not be assigned to the lineage. The viability of cells migrating from explants was assessed by DNA isolation and electrophoresis on days 6-16 of gestation. At all times evidence for apoptosis was found, even after culture intervals as brief as 4 h. Parallel analyses of histological sections of the metrial gland, using terminal deoxytransferase labelling to detect nuclear fragmentation, did not support significant levels of uNK cell death in situ prior to day 12 of gestation. Supplementation of the explant culture medium with estrogen, IL-2, various extracellular matrices, decidual cells or combinations of these did not lead to in vitro proliferation of uNK cells and usually did not extend the short term viability of these cells in serum supplemented or serum free media. Thus, the optimal culture conditions for uNK cells remain undefined.
