| 1. |
- Brand, Bodo, et al.
(författare)
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Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score
- 2011
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Ingår i: Genetics Selection Evolution. - London, UK : BioMed Central. - 0999-193X .- 1297-9686. ; 43:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: During the past ten years many quantitative trait loci (QTL) affecting mastitis incidence and mastitis related traits like somatic cell score (SCS) were identified in cattle. However, little is known about the molecular architecture of QTL affecting mastitis susceptibility and the underlying physiological mechanisms and genes causing mastitis susceptibility. Here, a genome-wide expression analysis was conducted to analyze molecular mechanisms of mastitis susceptibility that are affected by a specific QTL for SCS on Bos taurus autosome 18 (BTA18). Thereby, some first insights were sought into the genetically determined mechanisms of mammary gland epithelial cells influencing the course of infection.Methods: Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows selected for high and low mastitis susceptibility by applying a marker-assisted selection strategy considering QTL and molecular marker information of a confirmed QTL for SCS in the telomeric region of BTA18. The cells were cultured and subsequently inoculated with heat-inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 h, the cells were harvested and analyzed using the microarray expression chip technology to identify differences in mRNA expression profiles attributed to genetic predisposition, inoculation and cell culture.Results: Comparative analysis of co-expression profiles clearly showed a faster and stronger response after pathogen challenge in pbMEC from less susceptible animals that inherited the favorable QTL allele 'Q' than in pbMEC from more susceptible animals that inherited the unfavorable QTL allele 'q'. Furthermore, the results highlighted RELB as a functional and positional candidate gene and related non-canonical Nf-kappaB signaling as a functional mechanism affected by the QTL. However, in both groups, inoculation resulted in up-regulation of genes associated with the Ingenuity pathways 'dendritic cell maturation' and 'acute phase response signaling', whereas cell culture affected biological processes involved in 'cellular development'.Conclusions: The results indicate that the complex expression profiling of pathogen challenged pbMEC sampled from cows inheriting alternative QTL alleles is suitable to study genetically determined molecular mechanisms of mastitis susceptibility in mammary epithelial cells in vitro and to highlight the most likely functional pathways and candidate genes underlying the QTL effect.
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| 2. |
- Hartmann, Anja, et al.
(författare)
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Effects of threshold choice on the results of gene expression profiling, using microarray analysis, in a model feeding experiment with rat
- 2009
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Ingår i: Archiv für Tierzucht. - Dummerstorf, Germany : Research Institute for the Biology of Farm Animals (FBN). - 0003-9438. ; 52:1, s. 65-78
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Tidskriftsartikel (refereegranskat)abstract
- Global gene expression studies using microarray technology are widely employed to identify biological processes which are influenced by a treatment e.g. a specific diet. Affected processes are characterized by a significant enrichment of differentially expressed genes (functional annotation analysis). However, different choices of statistical thresholds to select candidates for differential expression will alter the resulting candidates list. This study was conducted to investigate the effect of applying a False Discovery Rate (FDR) correction and different fold change thresholds in statistical analysis of microarray data on diet-affected biological processes based on a significantly increased proportion of differentially expressed genes. In a model feeding experiment with rats fed genetically modified food additives, animals received a supplement of either lyophilized inactivated recombinant VP60 baculovirus (rBV-VP60) or lyophilized inactivated wild type baculovirus (wtBV). Comparative expression profiling was done in spleen, liver and small intestine mucosa. We demonstrated the extent to which threshold choice can affect the biological processes identified as significantly regulated and thus the conclusion drawn from the microarray data. In our study, the combined application of a moderate fold change threshold (FC≥1.5) and a stringent FDR threshold (q≤0.05) exhibited high reliability of biological processes identified as differentially regulated. The application of a stringent FDR threshold of q≤0.05 seems to be an essential prerequisite to reduce considerably the number of false positives. Microarray results of selected differentially expressed molecules were validated successfully by using real-time RT-PCR.
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