The identification of positive chicken factors for influenza polymerase activity
Ontology highlight
ABSTRACT: Using a chicken-hamster radiation hybrid panel (ChickRH6), we have mapped chicken chromosome(s) that contain possible factor(s) that permit avian polymerase activity, by increasing polymerase activity. We identified four hybrid clones permissive for polymerase activity. Activity was lost following 12 passages respectively in only one of the positive clones. Expression from the four positive radiation hybrid clones at passages 1, and 12 was measured and compared to expression of the hamster recipient line and 17 negative (for polymerase activity) clones.
Project description:Using a human-hamster radiation hybrid panel (G3), we have mapped loci regulating gene expression due to copy number increase. Copy number from radiation hybrid clones were measured relative to the A23 hamster recipient line. Expression from 79 radiation hybrid clones were measured in duplicate and compared to expression of the A23 hamster recipient line (GA_illumina_expression1 and GA_illumina_expression2). Expression of liver, kidney and heart from human and hamster are also included (GA_illumina_expression_R).
Project description:The heterotransplantation of human tumors to immune-deprived rodents has become an important preclinical tool for evaluating human tumor biology and responsiveness to therapeutic agents, based on the supposition that they reflect their original human origin. However, we demonstrate that such tumors can in fact change genetically, either by becoming tumors of the animal recipient or by becoming a hybrid of the human tumor and the animal hostâs cells. Three human tumors transplanted to the cheek pouch of golden hamsters resulted in permanently transplantable tumors that metastasize in the hamsters. Based on diverse lines of evidence, these transplants from a human glioblastoma multiforme and two Hodgkin lymphomas were malignant hybrids, showing both human and hamster genes in the malignant tumor cells, while retaining the morphology of the original human tumors. We demonstrate that even after being preserved for over 40 years in paraffin blocks, microarray analysis of RNA from these transplants disclosed over 3000 human genes derived from all 23 human chromosome pairs amongst these tumor transplants. A total of 3759 probe sets (ranging from 1040 to 1303 in each transplant) detected human gene transcripts in formalin-fixed, paraffin-embedded sections of the 3 hybrid tumors stored for over 40 years; the probe sets unambiguously map to 3107 unique Human Entrez Gene IDs and are representative of all human chromosomes, although, by karyology, one of the hybrid tumors (GB-749) had a total of 15 human chromosomes in its cells. Among the genes mapped, 39 probe sets, representing transcripts from 33 human genes , were detected in all hybrid tumor samples. Five of these 33 genes encode transcription factors that regulate cell growth and differentiation, five encode cell adhesion and transmigration-associated proteins known to participate in oncogenesis and/or metastasis and invasion, and additional genes encode components of pathways involved with signal transduction, regulation of apoptosis, DNA repair, and multidrug resistance. We posit that in-vivo fusion may disclose genes implicated in tumor progression, as well as gene families coding for the organoid phenotype. Thus, cancer cells can transduce adjacent stromal cells, with the resulting progeny having permanently transcribed genes with malignant and other functions of the donor DNA. Human gene expression profiles were determined by microarray analysis for 3 different Human-Hamster hybrid tumors, two Hodgkin lymphomas (GW-532, GW-584) and a glioblastoma multiforme (GB-749), that were first generated in the hamster cheek pouch after human tumor grafting and then propagated in hamsters and in cell cultures for years. Human gene expression was assessed using total RNA isolated from FFPE samples from GW-532 generations 2 and 34, GW-584 generation 28, and GB-749, in comparison to that for a Hamster control RNA sample isolated from a Hamster melanoma cell line (ATCC CCL-49). Expressed human genes were identified using MAS 5.0 signal expression values and detection P-values in the following manner: 1) Unannotated probe sets, as well as probe sets with no signal value greater than the median signal for AFFX spike-in controls with all Absent Detection Calls, were omitted from further analysis; 2) All remaining signal values for the hamster cell line sample were multiplied by the ratio of the median signal in all FFPE hybrid samples for AFFX spike-in control probe sets called present in all samples divided by the median signal for the same probe sets in the hamster CCL-49 sample; 3) Human transcripts were considered positive in a human-hamster hybrid FFPE sample if (a) a probe set signal exhibited a 2-fold or greater increase in any FFPE hybrid sample compared to the CCL-49 coontrol sample, (b) the fold change was greater than 2 standard deviations for that probe set across the FFPE samples, and (c) was called present (P) or marginal (M) for at least one or more FFPE samples.
Project description:Myofibroblast is a specific type of mesenchymal cell characterized by synthesis of extracellular matrix and contractile activity. While it serves a beneficial function during tissue wound healing under physiological conditions, it can cause devastating damage to organs afflicted with fibrosis. Myofibroblasts are also present in tumor stroma and contribute actively to tumor growth and spreading. Chicken embryo dermal myofibroblasts (CEDM) represent a novel ex vivo model suitable for the analysis of myofibroblastic phenotype as they show strongly pronounced, uniform and self-sustained myofibroblastic phenotype that is stable in time. As myofibroblastic differentiation is controlled chiefly by TGF-beta signaling, the understanding of the differentiation program entails the determination of TGF-beta-regulated genes. To achieve such a goal, we performed oligonucleotide microarray analysis of CEDM cells treated with a selective TGFBR1 kinase inhibitor. Genes reported previously to be under the control of TGF-beta signaling in mammalian cells appeared among the affected genes also in CEDM cells and many so far unknown TGF-beta targets were revealed. Comparison of the expression profiles of chicken embryo dermal myofibroblasts in culture treated with TGFBR1 Kinase Inhibitor II or DMSO only. Three biological replicates were analyzed for each group.
Project description:Two clones of Chinese hamster ovary (CHO) cells were cultured in 2 different chemical difined custom media. Transcriptional profile were compeared between clones and media.
Project description:Recombinant chicken IFN1 was prepared as previously reported (Laidlaw et al, 2013) and was added in chicken embryo fibroblasts to a final concentration of 1000 U/ml. Confluent cells were treated with chicken IFNα or mock treated and incubated for six hours before harvesting. The experiment was repeated in triplicate with three different batches of CEFs.
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:To examine global gene expression profile of chicken early paraxial mesoderm differentiation, we microdissected stage 12HH chicken PSM regions into 20 pieces (10 pieces both left-right PSM), including the tail bud, the PSM and somites. We create microarray series using these fragments. Duplicated 10 fragmented tissues from stage 12 chicken PSM regions. contributor: IGBMC microarray facility
Project description:To realize the gene expression in response to acute heat stress in chicken testis, we have employed whole genome microarray expression profiling as we have employed whole genome microarray expression profiling as a tool to identify genes response to acute heat stress. Male B strain Taiwan country chickens were subjected to acute heat stress (38M-bM-^DM-^C) for 4 h, and then exposed to 25M-bM-^DM-^C, with testes collected 0, 2, and 6 h after the cessation of heat stress, using non heat-stressed roosters as a control group (n = 3 roosters per group). Based on a chicken 44K oligo microarray, 163 genes significantly differed in the testes of the heat-stressed chickens from those of the control chickens. The mRNA expressions of upregulated genes, including HSP25, HSP90AA1, HSPA2, and LPAR2, and downregulated genes, including CDH5, CTNNA3, EHF, CIRBP, SLA, and NTF3, were confirmed through quantitative real-time polymerase chain reaction (qRT-PCR). Acute heat stress induced testicular gene experssion in B strain Taiwan country chicken was measured at 0, 2, and 6 h of recovery after 4 h of 38 degree acute heat stress.
Project description:Using Immunoprecipitation (IP) and mass spectrometry analysis, we aimed at identifying the target of a self-made monoclonal antibody to an unknown molecule on the outer membranes of chicken peripheral blood derived mononuclear cells (PBMC).
Project description:Recombinant chicken IFN1 was prepared as previously reported (Laidlaw et al, 2013) and was added in chicken embryo fibroblasts to a final concentration of 1000 U/ml. Confluent cells were treated with chicken IFNa or mock treated and incubated for six hours before harvesting. The experiment was repeated in triplicate with three different batches of CEFs.