The sensitivity, specificity and repeatability of the assay were validated by testing semen samples from 12 boars inoculated experimentally with PCV2, 10 boars infected naturally with PCV2, and 3 PCV2 negative control boars. The duplex qPCR assay was found to be more sensitive, specific, rapid, and repeatable than nested Avapritinib ic50 PCR (nPCR) methods for
the detection of PCV2 DNA in semen. Analysis of separated semen fractions by the duplex qPCR assay showed PCV2 DNA to be present mainly in the cell fraction as opposed to the seminal plasma fraction which is in contrast to previous reports. The duplex qPCR assay was found to be a valuable too] for accurate and quantitative detection of PCV2 DNA in boar semen. (c) 2008 Elsevier B.V. All rights reserved.”
“Basal forebrain neurons express the neurotrophin receptors, p75NTR and tyrosine S63845 ic50 kinase receptor A (TrkA). We tested the hypothesis that impairment of memory in rats could be achieved by RNA interference
(RNAi) -induced silencing of TrkA specifically within these neurons. A novel fusogenic, karyophilic immunoporter (fkAb(p75)-ipr) was constructed from the antibody, MC192 (monoclonal antibody to the rat neurotrophin receptor p75NTR, Ab(p75)), poly-L-lysine together with the hemagglutinin 2 and VP1 nuclear localization peptides of influenza and SV40 virus, respectively. Plasmid DNA constructs containing Dipeptidyl peptidase short hairpin sequences inhibitory to tyrosine kinase receptor A expression (TrkAi) and the gene encoding cGFP (green fluorescent protein from coral fish) was produced. These TrkAi plasmids were mixed with the immunoporter, forming the immunogene, TrkAi-fk-Ab(p75). A control TrkAsc complexed with fkAb(p75) (TrkAsc-fkwAb(p75)) immunogene was constructed from a scrambled sequence (TrkAsc) and fkAb(p75-ipr). Rats were infused using an osmotic mini-pump into the third ventricle with either TrkAi-fkAb(p75) or TrkAsc-fkAb(p75). Naive rats were also
included as additional controls. After 7 days, examination of gene expression on forebrain sections of some rats revealed cGFP expression in TrkA neurons. Fifteen to 19 days after infusion, rats were tested in a Morris water maze apparatus. Animals that received TrkAi-fkAb(p75) showed significantly impaired spatial memory learning ability compared with naive or TrkAsc-fkAb(p75)-treated rats. Western blot and immunofluorescence analysis showed that TrkA protein levels and numbers of TrkA positive neurons were reduced by 60% and 55% respectively in TrkAi-fkAb(p75)-infused rats compared with infused controls or naive animals. We conclude that p75-receptor-mediated RNAi-induced silencing of genes offers a novel and powerful way to study the function of specific endogenous genes within distinct neuronal subpopulations of the brain.