Project description:Hesx1+/+ and Hesx1-/- ES cells were cultured in 2i/LIF conditions for 5 passages, to induce a ground pluripotency state. Subsequently, inhibitors were removed to stimulate exit from ground pluripotency, and whole transcriptomics analysis was performed 24 hours after removal.
Project description:Synthetic oligonucleotides (ODNs) containing CpG motifs stimulate human plasmacytoid dendritic cells (pDCs) to produce type-1 interferons (IFN) and pro-inflammatory cytokines. Previous studies demonstrated that interferon regulatory factors (IRFs) played a central role in mediating CpG-induced pDC activation. This work explores the inverse effects of IRF-5 and IRF-8 on CpG dependent gene expression. Results from RNA interference and microarray studies indicate that IRF-5 up-regulates TLR9-driven gene expression whereas IRF-8 down-regulates the same genes. Several findings support the conclusion that IRF-8 inhibits TLR9 dependent gene expression by directly blocking the activity of IRF-5. First, the inhibitory activity of IRF-8 is only observed when IRF-5 is present. Second, proximity ligation analysis shows that IRF-8 and IRF-5 co-localize within the cytoplasm of resting human pDC and co-translocate to the nucleus after CpG stimulation. Taken together, these findings suggest that two transcription factors with opposing functions control TLR9 signaling in human pDCs. CAL-1 cells were transfected with siRNA targeting IRF-5 (IRF-5si) and left unstimulated (n=4, technical repeats) or stimulated with K-type CpG ODN (n=4, technical repeats). CAL-1 cells were transfected with siRNA targeting IRF-8 (IRF-8si) and left unstimulated (n=4, technical repeats) or stimulated with K-type CpG ODN (n=4, technical repeats). CAL-1 cells were transfected with control siRNA (Contsi) and left unstimulated (n=4, technical repeats) or stimulated with K-type CpG ODN (n=4, technical repeats).
Project description:DEAD-box proteins, a family of RNA-dependent ATPases, promote the numerous conformational rearrangements required for spliceosome assembly, activation, and disassembly. Previous work showed that a cold-sensitive substitution in DEAD-box protein Prp28 prevents the switch from U1 to U6 snRNA pairing with the 5’ splice site. Little is known about how Prp28 is regulated, although U5 snRNP protein Prp8 is a potential coordinator of Prp28 and other spliceosomal ATPases. We conducted a targeted selection in Prp8 for cold-insensitive suppressors of prp28-1, then used splicing specific microarrays to assess suppression of the prp28-1 splicing defect. Splicing specific microarrays were used to assess suppression of the prp28-1 splicing defect by a prp8 allele (prp8-tes) that suppresses prp28-1 cold-sensitivity
Project description:Transcriptional profiles on different yeast strain mutants (DEgd2/1, DEgd2/Btt1)were identified by microarray analysis comparing total mutant RNA vs wild type RNA. Set of arrays that are part of repeated experiments
Project description:Nascent polypeptides emerging from the ribosome during biogenesis can interact with many chaperones and protein homeostasis factors. The high sensitivity of RNA identification can be used to identify substrates of specific cotranslationally acting chaperones. Protein A-tagged (TAP-tag) chaperones associated with translating ribosomes were immunopurified by binding to magnetic IgG beads, washed, and chaperone complexes were eluted with TEV protease treatment. Immunopurified RNAs and total cell extract RNAs were isolated, reverse transcribed, coupled to Cy5 and Cy3 dyes, respectively, and comparatively hybridized to DNA microarrays Set of arrays that are part of repeated experiments
Project description:IL-17RA knockout mice presented increased tissue parasitism during T. cruzi infection that correlated with a reduced frequency of parasite-specific CD8+ T cells. Parasite-specific CD8+ T cells from KO mice showed similar proliferation rate but increased mortality and an exhausted phenotype when compared to WT counterparts. We hypothesize that during T. cruzi infection, IL-17RA signaling in CD8+ T cells is required for the regulation of a transcriptional program that prevents accelerated mortality and exhaustion and sustain the development of robust cytotoxic response required for efficient parasite control.
Project description:The present study reports the genetic and biochemical characterization of a dominant glossy mutant allele (BnaA. GL) in B. napus that results in a glossy phenotype. Results from transmission electron microscopy and scanning electron microscopy revealed the GL mutant exhibits reduced deposition of the cuticle layer, which was confirmed by a cuticular wax analysis. The wax compositional analysis revealed an increase in aldehydes but a severe decrease in alkanes, ketones and secondary alcohols. Genetic mapping narrowed the BnaA. GL gene to the end of A9 chromosome, where a gene homologous to ECERIFERUM1 (CER1) in Arabidopsis locates.<br><br>Then, we conducted a microarray analysis to find the differentially expressed genes between normal phenotype and glossy plants. Two comparisons were performed: wild type parent VS. the GL parent, and the bulked normal phenotype DH lines VS. the bulked glossy DH lines. The DH lines are generated from F1 plants of two parents, and RNA samples from three DH lines were combined to make a bulked sample for each phenotype. <br><br>Although no discernible mutation was apparent in the B. napus gene, this cDNA microarray chip assay revealed coordinated down regulation of genes encoding enzymes of the cuticular wax biosynthetic in the glossy mutant with BnCER1 being one of the most severely suppressed genes.
Project description:The overall gene expression profiles revealed by this system provide novel insight into how pluripotency is acquired in germ-committed cells. In the study presented here, we employed FACS sorting to purify pluripotent candidate cells during the culture period