Project description:Mycoplasma gallisepticum transcriptome comparison between in vitro grown cultures of strains Rlow and F utilizing oligo DNA microarrays.
Project description:Mycoplasma gallisepticum is a convenient model object for studying the regulation of transcription because it has a reduced genome, lack of cell wall and many metabolic pathways, and also easy to culture and non-pathogenic to humans. For rapid investigation of gene expression we developed microarray design including 3 366 probes for 678 genes. They included 665 protein coding sequences and 13 antisense RNAs from 816 genes and 17 ncRNAs present in Mycoplasma gallisepticum. This work was carried out transcriptomic profiling for different types of effects on the expression of genes of Mycoplasma gallisepticum: 1) genetic knock-out mutants; 2) cell culture exposed to sublethal concentrations of antibiotics; and 3) well-characterized heat stress effect. The study was performed on Agilent one-color microarray with custom design and random-T7 polymerase primer for cDNA synthesis. Using set of different probes for each gene or ncRNA allows to increase accuracy of gene expression quality.
Project description:To identify differential gene expression profiles of chicken tracheal epithelial cells (TECs) upon exposure to Mycoplasma gallisepticum virulent strain Rlow and avirulent strain Rhigh and corresponding lipid associated membrane proteins(LAMP) at 1.5 hours in vitro. Goal of this experiment was to identify relative comtribution of LAMPs in up-regulation of inflammatory gene compared to the live strains. Several genes were identified to be differentially regulated in all exposures, but the virulent strain up-regulated more number genes as well as at a higher extent. We identified 6 important inflammatory mediators and did confirmatory RT-qPCR analysis at 1.5, 6 and 24 hours in vitro as well as at 1.5 and 6 hours ex-vivo. RT-qPCR was also employed to identify expression of these 6 genes in presence of different signalling inhibitors and we were able to identify that Mycoplasma gallisepticum LAMPs up-regulate these inflammatory genes via TLR-2 in an NF-M-NM-:B dependent pathway. Primary chicken tracheal epithelial cells (TECs) were exposed to either 500 MOI of a virulent Mycoplasma gallisepticum strain Rlow or an avirulent strain Rhigh and the corresponding lipid associated membrane proteins (LAMPs) at 5M-BM-5g/mL for 1.5 hours. 4 biological replicates along with a dye swap technique totalling 8 replicates were utilized for all microarray experiments
Project description:Mycoplasma gallisepticum transcriptome comparison between in vitro grown cultures of strains Rlow and F utilizing oligo DNA microarrays. Two-condition experiment, Rlow vs. F strain cells. Biological replicates: 3. 1 technical replicate per biological replicate which includes a dye swap.
Project description:To identify differential gene expression profiles of chicken tracheal epithelial cells (TECs) upon exposure to Mycoplasma gallisepticum virulent strain Rlow and avirulent strain Rhigh and corresponding lipid associated membrane proteins(LAMP) at 1.5 hours in vitro. Goal of this experiment was to identify relative comtribution of LAMPs in up-regulation of inflammatory gene compared to the live strains. Several genes were identified to be differentially regulated in all exposures, but the virulent strain up-regulated more number genes as well as at a higher extent. We identified 6 important inflammatory mediators and did confirmatory RT-qPCR analysis at 1.5, 6 and 24 hours in vitro as well as at 1.5 and 6 hours ex-vivo. RT-qPCR was also employed to identify expression of these 6 genes in presence of different signalling inhibitors and we were able to identify that Mycoplasma gallisepticum LAMPs up-regulate these inflammatory genes via TLR-2 in an NF-κB dependent pathway.
Project description:Proteomes of Mycoplasma gallisepticum strains with overexpression and knockdown of WhiA transcription factor. The overexpression was introduced on a transposon vector carrying M. gallisepticum whiA gene with strong constitutive promoter and strong SD sequence. The knockdown was made by CRISPRi. dCas9 protein and sgRNA against whiA gene were introduced on a transposon vector.
