Project description:Hemagglutinin of the influenza virus is the main external glycoprotein. This very immunogenic protein is the target of the most anti-influenza vaccines. DNA vaccines are new alternative to conventional inactivated ones. Four DNA vaccines were tested. Each tested variant was based on the pCI vector with nucleotide sequence encoding hemagglutinin from A/swan/Poland/305-135V08/2006 (H5N1, clade 2.2). In K3/pCI, GK/pCI and HAneo/pCI the different optimization algorithms of hemagglutinin encoding sequence without amino acids change were tested. In 3NF/pCI the NFkappaB binding sites flanking the expression cassette were included in order to improve the nuclear transfer. Comparative transcriptome analysis of mice vaccinated the following vaccine HAneo/pCI,K3/pCI, GK/pCI or 3NF/pCI versus empty vector demonstrated minor changes in genes expression pattern. Most genes were expressed on the similar level in the vaccinated individuals and in the control mice. Small number of genes in particular variants showed the expression different than in the control mice. In general, the identified genes with the changed expression included some genes involved in metabolic processes and none of them seem to induce any undesirable pathways nor disease. Overall design: 6-8 weeks female mice BALB/c strain were used in the experiments. Three independent biological replicates of total RNA were isolated for each vaccine variants: HAneo/pCI, K3/pCI and 3NF/pCI and two independent biological replicates of total RNA were isolated from negative control group - empty vector and group GK/pCI. The spleen samples were collected 3 days after the booster DNA vaccination against influenza virus, each of dose was 20ug of plasmid DNA per mouse. Totally, 13 samples of RNA was prepared.
Project description:PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. Keywords: other
Project description:We collected whole genome testis expression data from hybrid zone mice. We integrated GWAS mapping of testis expression traits and low testis weight to gain insight into the genetic basis of hybrid male sterility. Gene expression was measured in whole testis from males aged 62-86 days. Samples include 190 first generation lab-bred male offspring of wild-caught mice from the Mus musculus musculus - M. m. domesticus hybrid zone.
Project description:We explored the relationship between the evolutionary dynamics of CTCF binding and the functional stability of higher order genome structures, by performing ChIP-seq experiments in closely related Mus species or strains and intersecting with Hi-C-derived topologically associating domains (TADs) and expression data. Experiments were performed in adult male liver samples, using input control sets.
Project description:The cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self renewing cell populations that constitute the bulk of the tumor. Targeting breast CSC (BCSC) self-renewal represents an avenue for developing therapeutics; however, the molecular mechanisms that govern self-renewal of BCSCs are poorly understood. Our data show the small molecule ID8 decreases overall cell growth, but increases the self-renewal of Aldefluor+ BCSCs and increases functional metastatic BCSCs in a xenograft model. Microarray analysis showed that ID8 is a pleotropic molecule by increasing numerous pathways, including cytokines and chemokines. However, inhibition of those pathways does not abrogate the ID8-induced increase in Aldefluor+ BCSCs. Rather, ID8 is able to activate MAPK pathway through upregulation of the scaffold protein LAMTOR3 and inhibition of MEK prevented the increase in Aldefluor+ BCSCs. By using ID8 as a molecular tool, we identified a new function of the MAPK pathway in regulating BCSC growth and self-renewal. Overall design: In three separate experiments per cell line, SUM159 and T47D breast cancer cells were treated with DMSO or 5uM (micromolar) ID8 for 3 days. DMSO and ID8 treated cells were trypsinized, processed, and stained for intracellular aldehyde dehydrogenase (ALDH) activity using the Aldefluor assay to identify cancer stem cells (Aldefluor+). CSCs and non-CSCs (Aldefluor-) were collected by flow cytometry from both groups. Total RNA was collected from each fraction (Aldefluor+, Aldefluor-), treatment (DMSO, ID8), and experiment (#2-7). A total of 24 samples were analyzed on an Affymetrix Human Gene ST 2.1 plate. Differential gene expression was determined with R/Bioconductor with Robust Multiarray Average (RMA) and fitting the data to linear models (limma). Experimental and processing batch effects were accounted for using ComBat. Eight sets of univariate comparisons were made: 1) SUM159 ID8 Aldefluor+ vs SUM159 DMSO Aldefluor+; 2) SUM159 ID8 Aldefluor+ vs SUM159 ID8 Aldefluor-; 3) SUM159 DMSO Aldefluor+ vs SUM159 DMSO Aldefluor-; 4) SUM159 ID8 Aldefluor- vs SUM159 DMSO Aldefluor-; 5) T47D ID8 Aldefluor+ vs T47D DMSO Aldefluor+; 6) T47D ID8 Aldefluor+ vs T47D ID8 Aldefluor-; 7) T47D DMSO Aldefluor+ vs T47D DMSO Aldefluor-; 8) T47D ID8 Aldefluor- vs T47D DMSO Aldefluor-. Multiple testing comparisons were adjusted using Benjamini and Hochberg (aka FDR). Probes with an adjusted p-value <0.05 were considered statistically significant.
Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes. Overall design: Mouse hematopoietic stem cells were purified from bone marrow cells using negative and positive selection with a Magnetic-Activated Cell Sorter (MACS). total RNA and mRNA were purified from the purified cells using Trizol reagent and magnetic oligo dT beads. Double strand cDNAs were synthesized using a cDNA synthesis kit and anchored oligo dT primers. After NlaIII digestion, 3’ cDNAs were isolated and amplified through 16-cycle PCR. SAGE tags were released from the 3’ cDNA after linker ligation. Ditags were formed, concatemerized and cloned into a pZERO vector. Sequencing reactions were performed with the ET sequencing terminator kit. Sequences were collected using a Megabase 1000 sequencer. SAGE tag sequences were extracted using SAGE 2000 software.
Project description:Investigating type I interferon in prediabetic stage of DCs from NOD mice compared to C57BL/6.g7 and Rag KO mice. Overall design: RNAseq was used to study splenic cDC1, cDC2, pDC, moDC, and monocyte populations from 8-10 week old NOD and C57BL/6.g7 and Rag KO mice.