A decade of using small-to-medium throughput allele discrimination assay to determine prion protein gene ( Prnp) genotypes in sheep in Slovenia.

Sheep with valine (V) at codon 136 and glutamine (Q) at codon 171 of the prion protein gene ( Prnp) are highly susceptible to classical scrapie, whereas phenylalanine (F) at codon 141 and histidine (H) at codon 154 play a major role in the susceptibility to atypical scrapie. A TaqMan real-time PCR assay was developed to determine Prnp alleles at codons 136, 141, 154, and 171 and used in classical scrapie eradication and breeding programs adopted in Slovenia. The frequency of the most resistant genotypes ARR/ARR and ARR/ARQ increased significantly in tested animals ( n = 35,138) from 6.7 and 27.1% of the tested sheep in 2006 to 12.1 and 32.4%, respectively, in 2015. Frequencies of more susceptible genotypes ARQ/ARQ and ARQ/VRQ decreased significantly from 36.4 and 3.5% in 2006 to 31.1 and 1.8%, respectively, in 2015. The most susceptible genotype VRQ/VRQ was detected in <0.5% of tested sheep. Frequencies of alleles AFRQ and AHQ affecting the susceptibility to atypical scrapie did not change significantly. The developed assay was suitable for genotyping on a small-to-medium throughput scale and was successfully used in classical scrapie eradication, as well as for the selection of classical scrapie-resistant sheep within breeding programs in Slovenia.

The number of cells expressing dopamine D2 receptor mRNA in rat brain caudate putamen is higher in oestrus.

Dopamine D2 receptors (D2-Rs) in the central nervous system are involved in the control of locomotion, cognition, emotion and neuroendocrine secretion. The intensity of cellular responses to specific stimuli is dependent on the concentration of dopamine or its agonist, and the availability, as well as the concentration, of all the other components of the signalling pathway in the cell, including the receptors. Many factors can influence the level of mRNA encoding the receptors. In order to study the changes in the level of expression of the D2-R mRNA in the brain of female rats at different stages of the oestrous cycle, we used a quantitative in situ hybridization technique. Four groups of animals were analysed: rats in prooestrus (POE), oestrus (OE), dioestrus 1 (DOE1) and dioestrus 2 (DOE2). A 35S-labelled riboprobe was transcribed in vitro from the 1.5-kb D2-R cDNA. The caudate putamen of the rats, which shows the highest level of expression of D2-R mRNA in the brain, was examined. The number of silver grains per cell, representing hybridization of riboprobe, and the number of cells expressing the D2-R mRNA, were counted with the computer-assisted image analysis system Lucia-M. Our results show that the expression of the D2-R mRNA in the lateral striatum varies during the oestrous cycle, with the highest expression measured during DOE2. The number of cells expressing the D2-R mRNA also changes during the different phases, with the highest number being detected in OE. This indicates that during OE more cells transcribe the D2-R mRNA. The results suggest that the variations in the concentration of the D2-R mRNA in the caudate putamen of the rat brain at different stages of the reproductive cycle are caused by the combination of variable expression of the mRNA per cell and different number of the cells that express the mRNA.