Expression profiling of DT40 chicken B cell line by RNA-seq
ABSTRACT: A single replicate of exponentially growing DT40 CL18 chicken B lymphoma cells were harvested and extracted RNA was subjected to Illumina GAIIx paired-end sequencing to determine global gene expression. Single replicate RNA-seq expression analysis of DT40 cells.
Project description:RNA-seq was performed for transcriptional analysis of wild-type DT40 cells (Gallus gallus, B-cell line) and a H3.3 knockout line (h3.3a-/-, h3.3b-/-). H3.3 is a H3 histone variant encoded on two genes (H3.3A and H3.3B) in chickens.
Project description:PAx5 is indispensible for the committment if early lymphoid progenitors to the B cell lineage as well as for the development and maintenance of B cells. To better understand the functional importance of Pax5 in the later stages of B cell development and investigate the targets of Pax5 regulation, we established a novel Pax5 deficient DT40 B cell line.
Project description:Transcription profiling of chicken Pax5 deficient DT40 B cell line to investigate the targets of Pax5 which is required for B-cell differentiation Used in cross-species comparison to investigate evolutionarily conserved regulatory circuits in B cell development Pax5 deficient DT40 B cells (3 biological replicates) were compared to DT40 wild-type cells (3 biological replicates).
Project description:The transcription factor Bcl6 is required for germinal center formation and deregulated expression of Bcl6 has been observed in lymphomas. To gain insight to the function of Bcl6 in terminal differentiation of B cells to plasma cells and to investigate the targets of Bcl6, we established a Bcl6 deficient DT40 B cell line.
Project description:To further elucidate the mechanism of bursal-derived bioactive peptides on avian B cell proliferation on the broad molecular level, we have employed whole genome microarray expression profiling as a discovery platform to examine gene expression patterns during BP5 and BSP-II treatment in DT40 cell culture system. Analysis of our expression microarray data in this manner defined 3163 genes up-regulated and 3539 genes down-regulated by BP5 treatment and 3117 genes up-regulated and 3531 genes down-regulated by BSP-II treatment at 95% confidence, with a commonly regulated gene set of 2253 upregulated genes and 2083 downregulated genes. Various pathways were significantly impacted by BP5 and BSP-II treatment, and gene Ontology annotations show changes in the expression of molecules involved in DT40 cell with BP5 and BSP-II treatment. Expression of 12 genes (FGF3, RAP1B, TCEB1, CSNK2A1, DNMT1, HIF1A, HDAC1, FZR1, GDF3, FGF8, IRF7, and SKP1) from this signature was quantified in the RNA samples by QRT-PCR, confirming low variability between the predicted response patterns. BP5 and BSP-II induced gene expressions in DT40 cell were measured at 4 hours after exposure to doses of 0.02ug/ml and 2ug/ml, respectively. Two independent experiments were performed using different cells for each experiment.Three independently generated populations of cells were used for these experiments.
Project description:PTIP (Pax2 transactivation domain-interacting protein) is a nuclear protein containing six BRCT domains. It has been shown that PTIP affects gene expression by controlling the activity of the transcription factor Pax2 and histone H3 lysine 4 methyltransferase complexes. In addition to its role in transcriptional regulation, PTIP has been implicated in DNA damage response. To ask if the depletion of PTIP affects the expression level of genes encoding DNA damage response factors , we compared the whole transcripts between wild-type and PTIP deficient chicken DT40 B cell lines. The total RNAs were isolated from wild-type and PTIP deficient cells (PTIP-/-/-) using Sepasol®-RNA I (Nacalai tesque, Japan). The gene expression profiles were examined using Genechip® Chicken Genome Array (Affymetrix Cat #900590), by GeneticLab co. Ltd. Japan, following Genechip® protocol.
Project description:Purpose: to identify how condensin I removal affects gene expression globally. Methods: Chicken DT40 CAP-H KO cells were treated with or without dox for 36 h. Total RNA samples were extracted and subjected to sequencing using an Illumina Hiseq2000 platform. Library preparation and Illumina sequencing were performed by Macrogen (South Korea). The sequence tags were spliced-mapped onto the chicken genome galGal4 using Tophat and Bowtie2 following quality test using FASTQC. Differential expression of genes was analyzed using the Bioconductor v2.3 package edgeR v3.2.3. Tag enrichment in NCBI RefSeq genes was calculated between dox-treated and untreated cells using edgeR exact test with tag-wise dispersion estimation. Results: We identified a total of 3798 genes to be differentially expressed in G1 condensin I-depleted chicken DT40 cells: 2495 down regulated and 1303 up regulated. Conclusions: Removal of CAP-H leads to significant misregulation of gene expression, suggesting a key role for condensin I in transcription during interphase. Total RNA profiles of CAP-H KO cells with or without Dox were generated by sequencing using Illumina Hiseq2000 platform.
Project description:In chicken DT40 cells, there are six linker histone H1 variants and 12 of coding genes. We have previously reported of 11 out of 12 H1 knock out DT40 cells (Takami et al., Genes to Cell 1997 [PMID:9491804]) but complete H1 null DT40 cells could not established, so far. We identified one of the H1 variant, H1R was involved in genomic instabilities (Hashimoto et al., DNA repair (2007) ), so we re-introduced floxed H1R-eGFP and mer-cre-mer into 11 out of 12 H1 knock out DT40 cells. Then we targeted last enedogenous H1, we successfully established conditional H1 KO cells (K11). Next we treated with tamoxifen to loop out floxed H1R-eGFP, and cloning H1 completely null cells (K11-5, and K11-7). We analysis those gene expression pattern in wild-type, K11, and K11-5 cells Experiment Overall Design: Apoptosis is induced in H1 null cells, so we inhibit apoptosis with pan-caspase inhibitor, Z-VAD-FMK and extract RNAs.
Project description:Global gene expression profiling of the avian B-lymphoma DT40 cell line was used as a model to differentiate among Btk KO and Btk KO cells reconstituted with human Btk. Differences in the gene expression pattern showed statistically significant changes between parental DT40 and all the Btk KO cell populations irrespective of whether they are reconstituted or not. These results imply that in the process of generating a knockout cell line, subclones are selected, which have multiple changes in their gene expression pattern (p<0.01). Experiment Overall Design: DT40 cells along with the mutants were grown at various time points in different batches of fetal calf serum with or without any stimulation. All experiments were repeated ten times and then polled together as one sample. In total 28 samples were used RNA extraction and hybridization on Affymetrix microarrays.