Synthesis, serum stability and cell uptake of cyclic and hairpin decoy Oligonucleotides for TCF/LEF and GLI transcription factors

A series of hairpin oligonucleotides bearing a 5?-alkyne and 3?-azide were cyclised using the CuAAC reaction to form cyclic decoys for targeting the DNA binding site of the TCF/LEF and GLI transcription factors. Incubation of a fluorescent hairpin oligomer and its cyclic analogue in fetal calf serum showed that the cyclic construct has significantly greater stability to enzymatic degradation. Cell uptake studies using HEK-293 cells with the fluorescent cyclic decoy in the presence of lipofectamine 2000 transfection agent indicated that this analogue is taken up by the cells and localizes to the cell nucleus. Localized fluorescence was observed in the nuclei of HEK-293 cells after only 1.5 h incubation which increased over a period of 4 h and persisted for 24 h

Evaluation of the Serotonergic Genes htr1A, htr1B, htr2A, and slc6A4 in Aggressive Behavior of Golden Retriever Dogs

Aggressive behavior displays a high heritability in our study group ofGolden Retriever dogs.Alterations in brain serotonin metabolism have been described in aggressive dogs before. Here, we evaluate whether four genes of the canine serotonergic system, coding for the serotonin receptors 1A, 1B, and 2A, and the serotonin transporter, could play a major role in aggression in Golden Retrievers. We performed mutation screens, linkage analysis, an association study, and a quantitative genetic analysis. There was no systematic difference between the coding DNA sequence of the candidate genes in aggressive and non-aggressiveGoldenRetrievers.An affecteds-only parametric linkage analysis revealed no strong major locus effect on human-directed aggression related to the candidate genes. An analysis of 41 single nucleotide polymorphisms (SNPs) in the 1 Mb regions flanking the genes in 49 unrelated human-directed aggressive and 49 unrelated nonaggressive dogs did not show association of SNP alleles, genotypes, or haplotypes with aggression at the candidate loci. We completed our analyses with a study of the effect of variation in the candidate genes on a collection of aggressionrelated phenotypicmeasures.The effects of the candidate gene haplotypes were estimated using the Restricted Maximum Likelihood method, with the haplotypes included as fixed effects in a linear animal model. We observed no effect of the candidate gene haplotypes on a range of aggression-related phenotypes, thus extending our conclusions to several types of aggressive behavior.We conclude that it is unlikely that these genes play a major role in the variation in aggression in the Golden Retrievers that we studied. Smaller phenotypic effects of these loci could not be ruled out with our sample size