F8 broiler by Leghorn and broiler by Fayoumi spleen at day 7 and 8 after challenge with SE
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ABSTRACT: In the two F8 advanced crosses of broiler by Leghorn and broiler by Fayoumi, birds at day 1 were challenged with Salmonella enteritidis (SE). Spleen were collected at day 7 and 8. SE bacterial load in spleen were measured. Based on the bacterial load, birds were divided into high and low SE load groups. At each line cross and each day time point, three pair comparisons among high SE load, low SE load, and non-SE were used for the loop design, and two biological replicates were used.
Project description:Necrotic enteritis is a disease caused by Clostridium perfringens, which threatens poultry production in the absence of dietary antibiotics. A total number of 144 Ross broilers were reared in 12 pens with each hosting 12 birds. Each 6 pens of birds were fed medicated (bacitracin at 55 ppm) or non-medicated starter diets (Nutreco Canada Agresearch) immediately after the chicks were placed. At day 18, birds were challenged with C. perfringens (107 cfu per ml mixed with feed). Spleens were collected from 12 birds of each group at day 18 (before infection), 19, 20, and 22. A low-density chicken immune microarray was used to study gene expression profiling of host response to C. perfringens infection. Six biological replicates (2 birds per biological replicate) for each treatment group were labeled with either Cy5 or Cy3 with dye swap. A total of 24 arrays were used for this study. Gene signal intensity was globally normalized by LOWESS and expressed as log2 ratios. A mixed model including treatment, time, array, subgrid (random effect), dye, and all interactions among treatment and time was used to identify differentially expressed genes between post-infection vs. pre-infection, among post-infections, and between medication treatments, at the 5% significance level. The results indicated subtle medication effects on gene expression of these immune-related genes compared to bacterial infection effect. Our findings strongly suggest that both cell-mediated and antibody-mediated immune responses via MHC class I and II systems were actively involved in the host defense against C. perfringens infection in broilers. The unique cytokine signaling pathway and apoptosis cascade found in the study provide a new insight of molecular regulation of host immune response. Collectively, the findings of the present study will shed light on the molecular mechanisms underlying C. perfringens infection in broilers. There were two groups: medicated and non-medicated. Spleen were collected to isolate total RNA for gene expression profiling.For the microarray study, two birds from each pen were pooled within each group. To account for any bias inherent to the fluorescent dyes, three of the six medicated replicates were labeled with Cy3 and the other three were labeled with Cy5 at each time point. The same design was applied for the non-medicated group. There were six hybridizations between medicated and non-medicated replicates at each time point. Twenty-three out of 24 arrays were used (data from one array were discarded due to poor quality).
Project description:Circadian rhythms are daily oscillations of multiple biological processes driven by endogenous clocks. Imbalance of these rhythms has been associated with cancerogenesis in humans. To further elucidate the role of the circadian clock in cellular growth control, tumor suppression and cancer treatment, a standard of circadian gene regulations in healthy men is essential. Comparative microarray analyses were conducted investigating the relative mRNA expression of clock genes throughout a 24-hour period in biopsies obtained from oral mucosa of eight healthy diurnally active male study participants. A custom-designed oligo-based microarray which in addition to oncogenes and inflammatory genes contains probes for 20 clock genes and 70 clock-associated genes in human was used. Keywords: Human gene expression study Reference design with Cy3 labeled uniRNA and Cy5 labeled sample RNA.
Project description:Mesenchymal stem cells (MSC) omplexed to different transfection agents (R60, R200, DOSPER, jetPei, Pulsin). Two MSC populations (h21 and h24) and two passages (P2 and P4) were tested with different labelings. In a direct design experiment each sample was compared to its unlabeled control sample. Keywords: Human gene expression study Reference design with unlabeled cells in the Cy3 reference channel, and labeled cells in the Cy5 channel.
Project description:Wound healing is associated with high rates of cell replication and lactate accumulation even under normoxic and hyperoxic conditions. Lactate accounts for various effects in tissue regeneration, such as collagen synthesis, angiogenesis, modulation of cytokine patterns and as recently shown for stem cell homing. Its influence on genes involved in cell replication has not been shown yet. Therefore, the effect of lactate considering genes involved in different cellular processes was investigated. Human umbilical vein endothelial cells (HUVEC) were cultured and incubated with lactate for different periods of time. Gene expression analysis was performed using custom-designed oligonucleotide microarrays. Keywords: Human gene expression study Reference design with Cy3 labeled uniRNA and Cy5 labeled sample RNA.
Project description:The impacts of maternal Cd on zebrafish from female to embryo which included fecundity, gametes development, growth, other reproductive functions, and gene expression of 5hpf embryo were up and down regulated after female pretreated with Cd. The mating rate decreased by 30 %. It can be observed in growth delay during 6-somite stage. The ceratohyal head angle of larvae is widest upon 35.6 M-NM-<M maternal Cd treatment and it was showed a dose response. Besides, pericardial edema occurred in some 96hpf larvae. There were showed 33 and 37 target genes appeared significantly down and up regulation after microarray assay, respectively. The major effect on gene up regulation was showed at the functional of transcription (occupied 18.9%) and down regulation at the functional of protein biosynthesis (occupied 33.3%). These results demonstrated that maternal Cd influenced the reproduction ability of female and causes development abnormal of embryo. Sexually mature zebrafish (D. rerio) of both sexes, obtained from the Institute of Cellular and Organismic Biology, Academia Sinica (Taipei, Taiwan), were kept in an aquarium supplied with dechlorinated, circulated, aerated local tap water at 28 M-BM-0C with a 14: 10-h light/dark cycle, and were fed Daphnia and shrimp. Fertilized eggs were incubated in M-bM-^@M-^\zebrafish solutionM-bM-^@M-^\ (E3 solution) which contained 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 1 ppm methylene blue (pH 7.2). Developing embryos of 5 h post-fertilization (hpf) were collected for total RNA extraction, and process of microarray assay. The number of hatched zebrafish larvae at 48 and 72 hpf were recorded. The larvae of 72 hpf larvae were collected for cartilage stain in order to observe the development of pharyngeal arch. Nguyen and Janssen (2001) reported that the LC50 at 96 h of Cd was 0.62~1.33 M-NM-<M for 12-dpf stage zebrafish, and our previous study showed that the zebrafish embryo could induced physiological response with 0.89 M-NM-<M Cd exposure (Wu et al., 2008). Hence, a 10M-CM-^W (8.9 M-NM-<M) , 20M-CM-^W (17.8 M-NM-<M) and 40M-CM-^W (35.6 M-NM-<M) dose of 0.89 M-NM-<M was used in female zebrafish. The Cd medium was prepared using completely dried CdCl2 (Sigma, St. Louis, MO) dissolved in 1 mL concentrated HCl; double-deionized water was used to prepare the 10 mg/L Cd stock solution, which was then diluted with zebrafish solution before being used in the exposure experiments. During all experiments, the test containers were cleaned with HNO3 and thoroughly rinsed with double-deionized water before use.
Project description:Series containes 4 independent experiments and high and low power scanns for each independent experiment. Genome-wide mRNA expression profiles of Saccharomyces cerevisiae growing under hydrostatic pressure were characterized. We selected a hydrostatic pressure of 30 MPa at 25°C because yeast cells were able to grow under these conditions, while cell size and complexity were increased after decompression. Functional characterization of pressure-induced genes suggests that genes involved in protein metabolism and membrane metabolism were induced. The response to 30 MPa was significantly different from that observed under lethal conditions because protein degradation was not activated under 30 MPa pressure. Strongly induced genes included those that contribute to membrane metabolism and which are also induced by detergents, oils, and membrane stabilizers.
Project description:A comprehensive search for downstream genes is necessary for understanding of functions of Nodal signaling in embryos of the ascidian Ciona intestinalis. The level of mRNA expression in Nodal-overexpressing embryos was compared with that in normal embryos at the late gastrula stage by a microarray analysis. Overexpression of Ci-nodal elevated the level of expression of 115 genes more than two-fold. These candidate target genes include those encoding transcription factors, proteins involved in cell-cell communication, and extracellular matrix components. Many of these genes were expressed in the neural plate at the late gastrula stage. Overexpression of nodal also affected the expression of genes involved in the planar cell polarity pathway and Wnt/Ca2+ pathway (prickle, Rho-like2, and PLC2), and delta1-protocadherin-like. Keywords: ascidian, Ciona intestinalis, Nodal, embryogenesis, gene expression profile two samples Dye Swap design with 2 arrays
Project description:Recent evidences have demonstrated phosphatidylinositol3-kinase (PI3-K)/Akt signaling pathway participates in cell growth, cell survival, and adipocyte differentiation in vitro. However, the in vivo evidence to support Akt function on adipogenesis is limited. To achieve this goal, we generated the stable zebrafish transgenics of Tg(krt4:myrAkt1)cy18 carrying human constitutive active form of Akt1 (myrAkt1) that driven by a skin-specific krt4 promoter. The Tg(krt4:myrAkt1)cy18 display severely skin hypertrophy at embryonic stages, and support Akt1 function as a key gene on cell size control. When transgenics reached sexual maturation, they display obese phenotype due to adipocyte hypertrophy and up-regulation of adipogenesis-related genes. Collectively, our findings provided a direct evidence to support Akt play provital roles on cell size control as well as adipogenesis in vivo. In addition, the obese zebrafish line of Tg(krt4:myrAkt1)cy18 provide a new platform to study obesity and its related chronic diseases mechanism in vivo. The AB strain of Zebrafish (Danio rerio) were obtained from ZIRC (http://zebrafish.org/zirc/home/guide.php), and kept in the stock of Chung Yuan Christian University. Zebrafish were raised in a local tap water at 28.0±0.5℃ and under a constant photoperiodism of 14 hour light cycle/10 hour dark cycle. Zebrafish embryos were collected in 10 cm diameter Petri dishes containing 20 mL fish water after spawning and raised at 28.0±0.5℃. To prevent the disease of Zebrafish embryo, few drops methylen blue were added into fish water. At 5 to 7 dpf, larvae were transferred to 10 liter tanks containing 8.0 liter of fish water and fed with living Paramecium. After 14 dpf, larvae were feed with living artemia (OSI, USA) until adulthood. We dissected two tail tissues (as a pooled sample) from six month-old wild-type and Tg(krt4:myrAkt1)cy18 (n=5), and homogenized in Trizol reagent (Ambion) to isolate total RNA according to the manufacture instruction. The RNA was treated with DNase I at 25℃ for 10 minutes to remove DNA contamination and then cleanup by RNase-free spin columns (Qiagen). The total RNA concentration was determined by spectrophotometry (ND-1000 ; NanoDrop Technol, Wilmington, DE), and the RNA quality was checked by running electrophoresis in RNA-denatured gels. All total RNA were stored at -20℃. For real-time quantitative RT-PCR, 1μg of total RNA were reverse-transcribed with reverse transcriptase and the excess RNA were digested E.coli RNase H to enhance the cDNA purity. The commercial Zebrafish 14K oligo microarray chip was obtained from Institute of Cellular and Organismic Biology at Academia Sinica and contained 14067 oligonucleotides represent 9666 unique genes with a redundancy of 31%. The detail oligonucleotide description can be obtained from Ocimun Biosolution (http://www.ocimumbio.com/web/default.asp). We used SuperScriptTM Indirect cDNA Labeling System kit (Invitrogen) to generate the fluorescently labeled probes. Total RNA isolated from WT and Tg(krt4:myrAkt1)cy18 were reversed transcribed into cDNA and coupling for Alexa Fluor 555 and Alexa Fluor 647 fluorescent dyes (Invitrogen), respectively. Before hybridization, the Zebrafish microarray chips were pretreated with 1% bovine serum albumin, 4X SCC, and 1% sodium dodecylsulfate (SDS) for 45min at 42℃, and then hybridized in SlideHybTM buffer (Ambion) for overnight at 42℃. After hybridization, chips were washed with 2X SSC and 0.5% SDS for 15min at 25℃ and finally with 0.5X SSC and 0.5% SDS for 15min at 25℃. The fluorescence intensities of Alexa Fluor 555 and Alexa Fluor 647 targets were scanned by Genepix scanner (Molecular Devices, Sunnyvale, CA, USA) and the acquired date were analyzed by Genepix and Genespring software (Aglient Technologies, Foster City, CA, USA).
Project description:3 genetic chicken lines (Leghorn G-B1, Fayoumi, broiler) were used. chicken were challenged with SE at day 1, spleen and cecum tissues were collected at 2 hours and 16 hours after post challenge
Project description:To determine the transcriptional response to Salmonella enterica serovar Enteritidis (SE) infection, a newly developed chicken 44K Agilent array was used to analyze total RNA of heterophils from SE-resistant (line A) and SEâsusceptible chickens (line B), treated with in vitro infection of SE (I) or non SE medium (N) for 1 hr. A dual-color balanced design was used to provide a direct comparison between SE-infected and non-infected groups (AI/AN, BI/BN) and between line A and line B (AN/BN, AI/BI). Data retrieved from 426 immunologically important genes were used for functional analysis. The results indicated that: In the comparisons of SE infection with non-infection, 39 genes were found differentially expressed (P < 0.05 with at least 2-fold) after Salmonella infection, while 21 genes were found differentially expressed between different lineages. Numerous of members in Toll-like receptor (TLR) signaling pathway were identified which include receptors: MD-2, TLR-4, -5, -15; adaptors: TLR adaptor molecule1 (TICAM1/TRIF), MAP kinase kinase 3 (MKK3) and NFkB-1, as well as cytokines, chemokines and costimulatory molecule: interleukin (IL)-1β, IL-6, IL-8, IL-12, chemokine (C-X-C motif) ligand 1, chemokine (C-C motif) ligand 4, CCL 5, and CD80. Most of immune genes were up-regulated after SE infection, and the magnitude of up-regulation was higher in line A than line B in general. The results suggested that a similar TLR regulatory network exists in both lines with strong response in resistant line. This finding has laid the foundation for further study of molecular and cellular modulation of SE infection in chickens. Keywords: disease state analysis A dual color, balanced design was carried on for all of heterophils samples from sixteen chickens. Each sample type (AI, AN, BI and BN) includes four biological replication for labeling. A Dye swap was used in each pair of comparisons including AI/AN, BI/BN, and AI/BI, and AN/BN. Background subtracted signal intensity were collected from 16 arrays and normalized for data analysis.