Project description:Chickens divergently selected for either high abdominal fat content (fat genotype) or low abdominal fat content (lean genotype) at SRA-INRA, France were used to profile pituitary gland gene expression during juvenile development (1 to 7 weeks of age) and to identify differentially expressed genes associated with genotype and age. The fat line (FL) and lean line (LL) chickens are different in various phenotypic and metabolic measurements, including abdominal fatness, plasma glycemia, and T3. The FL and LL chickens represent unique models for characterizing biomedical and agricultural traits. The Del-Mar 14K Chicken Integrated Systems microarrays were used for a transcriptional scan in the pituitary gland during juvenile development using a reference design for the hybridization of total RNA from the pituitary gland. A reference RNA pool was made from an equal amount of high-quality total RNA extracted from all of the samples. Each of the microarrays was co-hybridized with Cy3-labeled cDNA targets from one of the pituitary samples and Cy5-labled cDNA targets from the reference pituitary RNA pool. Log2-transformed fluorescence intensities were analyzed with a mixed model. Two-way ANOVA (SAS) of log2 ratios was used to detect significant (p<0.05) differences by line, age, and the line-by-age interaction. There were 1150 significantly different genes between the two lines, and 339 of these genes exhibited greater than 0.68-fold differences in their log2 ratios (highest group mean at least 160% of the lowest group mean). One thousand four hundred twenty nine genes were significantly different by age and of these, 583 exhibited fold changes greater than 0.68 in the log2 ratio. There were 145 genes that significantly differed in their line-by-age interaction, and 62 of these exhibited greater than 0.68-fold differences. There were 386 genes with significant differences by line or for line-by-age interaction with at least a 0.68-fold change in log2 ratio and n ≥ 2 for each experimental group. Four biological replicates were analyzed, each consisting of a complete set of individual samples from pituitary glands of Fat and Lean line chickens at week 1, week 3, week 5, or week 7 of age. Amplified antisense RNA from each sample was analyzed using Del-Mar 14 K array in a reference design. Each experimental sample (Fat and Lean Weeks 1, 3, 5, and 7) was labeled with Cy3 and hybridized with an aliquot of the Cy5 reference pool, which was generated from all 32 RNA samples.

Project description:Genetic evidence has implicated several genes as being critical for heart development. However, the inducers of these genes as well as their other targets and the pathways they constitute, remain largely unknown. In the avian embryo, Cerberus (cCer) transcripts are detected in the anterior mesendoderm including the heart precursor cells and in the left lateral plate mesoderm. We have identified a promoter element of chick cCer able to drive EGFP expression into the heart and the hemangioblast precursor cells, allowing us to identify a population of cells that consistently exit from the anterior primitive streak region from as early as stage HH3+ and that later will populate the heart. In order to identify and study novel genes expressed and involved in the correct development and differentiation of the vertebrate H/HPC (Heart/ Hemangioblast Precursor Cells) lineages, a differential screening using Affymetrix GeneChip system technologies was performed. Remarkably, this screening led to the identification of more than 800 transcripts potentially expressed in these precursor lineages. We have identified unknown genes that are differentially expressed in the H/HPC precursor lineages. By developing a procedure to isolate the heart precursor cells using the cer2.5-EGFP construct, we were able to specifically isolate a population of H/HPC expressing already known cardiac markers, and a long list of still uncharacterized genes. From those, we defined by WISH that at least some of these in silico identified transcripts are in fact expressed in these cell populations and more importantly, functionally required for heart formation.More importantly, our study unveiled several uncharacterized genes that can now be used for further studies. Portions of chick embryos were selected and excised at early stages of development for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain homogeneous populations of embryos at early gastrula stage in order to increase the temporal resolution of expression profiles. To that end, we hand-selected and excised portions of the embryos according to morphological criteria and EGFP expression. Four GeneChip chicken arrays used for another study were added to the two arrays in this study to ensure a robust model for expression value computation (Note: The 4 samples chick_T2-1, chick_T2-2, chick_T2-3, chick_T2-4, were performed by another group and for a different array, and nothing has to do with our data. They were only run at the same time and used for normalization. Raw data unavailable.). The arrays were normalized to a baseline array with median CEL intensity by applying an Invariant Set Normalization Method. Normalized CEL intensities of the six arrays were used to obtain model-based gene expression indices (MBEI) based on a PM (Perfect Match)-only model. Processed data for all 6 samples can be found in supplementary file linked below.

Project description:We applied genomic array footprinting (GAF) in the search for genes of S. pneumoniae essential during the establishment and progression of experimental meningitis. Four libraries with a total of 6,000 independent TIGR4 marinerT7 transposon mutants were injected intra-cisternally in rabbits, and cerebrospinal fluid (CSF) was collected after three, nine, and fifteen hours. Microarray analysis of mutant-specific probes from CSF samples and inocula identified 82 genes of which mutants had become attenuated and eleven genes of which mutants had become enriched during infection. Screening results particularly point to an essential role for capsular polysaccharides (cps), nutrient uptake, and metabolism in meningitis. Detailed study on directed mutants of a subset of sixteen GAF targets in an experimental model of rat meningitis revealed that ten were significantly attenuated or enriched during competitive infection. Furthermore, we showed that mutants of adenylosuccinate synthetase (purA), flavodoxin (fld), and the substrate binding protein (livJ) of a branched chain amino acid ABC-transporter were essential for full blown meningitis in a mono-infection setup of rat meningitis. Overall, the GAF screen revealed the general restraint on nutrients encountered by the pneumococcus in CSF, while, except for cps genes, no ‘classic’ virulence factors appeared to be required for infection. This knowledge will contribute to a better understanding of the molecular pathogenesis of pneumococcal meningitis. The rabbit meningitis model was performed as previously described by Østergaard (PMID: 9661008 ). Briefly, 10E6 CFU of a S. pneumoniae mutant library was suspended in 20 µl beef-broth and injected into cisterna magna of four groups of four outbred New Zealand White rabbits of app. 2.5 kg (in one group one rabbit died). CSF (0.3 ml) samples were repeatedly obtained from the same rabbit by aspiration from indwelling spinal- and arterial cannules. Samples were collected before pleocytosis developed (three hrs), at the time of maximal pleocytosis (nine hrs), and at a time of slowing bacterial growth and abating pleocytosis (fifteen hrs ). Until further processing in the GAF protocol, CSF and inocula were stored with 15% glycerol at -80ºC. The GAF technology was performed as described previously (PMID: 17261526 ). Briefly, stored inocula and CSF obtained during the experimental rabbit meningitis experiments were defrosted, diluted in GM17 medium supplemented with spectinomycin, and grown to mid-log phase. Chromosomal DNA was isolated, digested with AluI endonuclease, and used as a template for in vitro transcription initiated from the T7 promoters of the marinerT7 transposon that is present in each mutant. After removal of template DNA by DNAseI treatment, mutant-specific T7 RNA was reverse transcribed in the presence of fluorescent Cy3/Cy5-labeled dUTP nucleotides. Labeled Cy3/Cy5-cDNA from the CSF samples and oppositely labeled Cy5/Cy3 cDNA from the inocula were combined, purified, and washed by ultrafiltration. The cDNA was hybridized to pneumococcal microarrays containing, 2,087 ORFs of Streptococcus pneumoniae TIGR4 and 70-mer oligo's specific for the non-homologous ORFs in the R6, D39, 23F, INV104B, INV200, OXC141, and G54 strains, all spotted in duplicate (PMID: 18174343) . Dual channel array images were acquired on a GenePix 4200AL microarray scanner (Axon Instruments, Union City, CA). Microarray image files were analyzed with GenePix Pro software (Axon Instruments, Union City, CA). Spots were screened visually to identify those of low quality and removed from the data set prior to analysis. A net mean intensity filter based on hybridization signals obtained with R6-specific spots was applied in all experiments. Slide data were processed and normalized using MicroPreP (PMID: 15907200). Further analysis was performed using a CyberT implementation of the Student’s t test (cybert.microarray.ics.uci.edu). This web-based program lists the ratios of all intra-replicates (duplicate spots) and inter-replicates (different slides), the mean ratios per gene, and standard deviations and (Bayesian) p values assigned to the mean ratios. For identification of conditionally essential genes, only genes with a minimum of 6/8 (for groups of four rabbits) or 5/6 for the group of three rabbits) reliable measurements and a Bayesian p-value < 0.001 were included. Further selection criteria were applied, as only mutants displaying an average fold-change of >2.5 at a minimum of two time-points, or >4.0 at one time-point were accepted as significantly attenuated or enriched. The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) database (www.ncbi.nlm.nih.gov/geo/) under GEO Series accession number ...

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Transcription profiling of two 24 hour old zebrafish embryos from the Tuebingen line

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