Application of a double-enrichment procedure for microsatellite isolation and the use of tailed primers for high throughput genotyping

The number of microsatellite loci and their allelic diversity contribute to increase accuracy and informativity of genetic estimates, however, the isolation of microsatellite loci is not only laborious but also quite expensive. We used (GATA)n and (GACA)n tetranucleotide probes and single- and double-enrichment hybridization to construct and screen a genomic library with an increased proportion of DNA fragments containing repeat motifs. Repeats were found using both types of hybridization but the double-enrichment procedure recovered sequences of which 100 percent contained (GATA)n and (GACA)n motifs. Microsatellite loci primers were then designed with an M13R-tail or CAG-tag to produce scorable PCR products with minimal stutter. The approach used in this study suggests that double-enrichment is a worthwhile strategy when isolating repeat motifs from eukaryotic genomes. Moreover, the use of tailed microsatellite primers provides increased resolution for compound microsatellite loci, with a significant decrease in costs.

Simultaneous overexpression of GH and STAT5b genes inhibits the STAT5 signalling pathway in tilapia (Oreochromis niloticus) embryos

In this study, we describe the use of a STAT5 responsive element (LHRE) reporter gene to monitor the activity of the growth hormone (GH) transduction pathway following expression of heterologous fish GH and rat STAT5b in tilapia embryos and fish fibroblast cells. Our results indicate that both GH and STAT5b are able to activate the LHRE at high levels when transferred separately, demonstrating the substantial level of conservation of the GH signal transduction pathways between fish and mammals. Unexpectedly, co-expression experiments show a strong inhibition of the GH-dependent activation, suggesting that simultaneous GH and STAT5b overexpression can counteract effects of GH expression in tilapia embryos