Gene expression profiling in HEK293 cells overexpressed lactoferrin gene that has the signal sequence (sLF) or not (ΔLF)
ABSTRACT: Human lactoferrin (LF) is a multifunctional protein involved in immunomodulation, cell growth, and differentiation. In addition to the secreted form (sLF), an alternatively spliced form (ΔLF) that lacks the signal sequence and downregulated in cancer was found. This study was carried out to identify and compare signaling networks provoked by the two LF isoforms. To do this, the two forms were overexpressed in HEK293 cells using the flp-in/tet-on system and genome-wide expression analysis of 18,367 genes was conducted. Secreted form (sLF) or alternatively spliced form (ΔLF) were overexpressed in HEK293 cells using the flp-in/tet-on system and genome-wide expression analysis of 18,367 genes was conducted.
Project description:Human lactoferrin (LF) is a multifunctional protein involved in immunomodulation, cell growth, and differentiation. In addition to the secreted form (sLF), an alternatively spliced form (ΔLF) that lacks the signal sequence and downregulated in cancer was found. This study was carried out to identify and compare signaling networks provoked by the two LF isoforms. To do this, the two forms were overexpressed in HEK293 cells using the flp-in/tet-on system and genome-wide expression analysis of 18,367 genes was conducted. Overall design: Secreted form (sLF) or alternatively spliced form (ΔLF) were overexpressed in HEK293 cells using the flp-in/tet-on system and genome-wide expression analysis of 18,367 genes was conducted.
Project description:Current study will help us to elucidate the differential gene expression profiles of two different subsets of monocytes stimulated with gamma-M. tuberculosis CD14hiCD16- and CD14loCD16+ monocytes were isolated from human peripheral blood using magnetic beads for CD14 and CD16. 1.5 million cells were cultured in 1 ml of complete RPMI medium and stimulated with 10 microgram/ml of gamma-M. tuberculosis for 48 hours. After 48 hours cells were harvested and RNA was isolated from control and stimulated samples from CD14hiCD16- and CD14loCD16+ monocytes. So total we had 4 samples: 1. Control CD14hiCD16- 2. Stimulated CD14hiCD16- 3. Control CD14loCD16+ monocytes and 4. CD14loCD16+ monocytes. RNA from all these above 4 samples were sent to Phalanx Biotech Group for whole genome microarray.
Project description:Microarray analysis of whole lung tissue from three mouse models of asthma Acute asthma was produced by immunization twice, one week apart, of Balb/C mice 8-12 weeks of age with Aspergillus species (5 μg) in adjuvant (1:1 vol/vol). Adjuvant was aluminum and magnesium hydroxide (Pierce). Asthma was initiated by three consecutive intranasal exposures to Aspergillus species (5μg in 15μL saline) and asthma was evaluated 72 hours after the final exposure. Tolerant asthma was produced by intranasal delivery of Aspergillus species (5μg in 15μL saline) twice a week for six consecutive weeks to Balb/C mice 8-12 weeks of age. Asthma was evaluated after mice were rested for an additional three weeks. Chronic asthma was produced by intranasal delivery of the Dustmite/ Ragweed/ Aspergillus (5,50,5ug in 15ul saline) mixture twice a week for six consecutive weeks in female mice Balb/C mice 8-12 weeks of age. Asthma was evaluated 3 weeks after cessation of allergen exposure. Saline control mice were treated intransally with 15ul of saline twice a week for 6 weeks. Asthma was assesed 3 weeks after the final exposure. n=3 samples/ mouse model.
Project description:Promoter methylation is able to induce downregulation of gene expression. 5-Aza-2'-deoxycytidine(Aza), methytransferase inhibitor, induce CpG demethylation. Here, 5-Aza-2'-deoxycytidine(Aza) is treated in a human breast cancer cell, MCF7, for detection of gene expression change. To analyze gene expression change by aza, control RNA isolated from MCF-7 was compared with RNA isolated from MCF-7 treated with 5uM and 10uM aza.
Project description:Investigating the effect of Tax protein on the expression of genes in Jurkat cells. Current study will help us to elucidate the differential gene expression profiles of Tax positive Jurkat cells. The Tax-positive (TaxP) and Tax negative (TaxN) subline of Jurkat were established. mRNA isolated from TaxN or TaxP cell lines were analyzed using gene array.
Project description:We explored the role of mammalian ETS1/2 and Mesp homologues of cardiogenic transcription factors of Ciona intestinalis, to convert primary human dermal fibroblasts into cardiac progenitors. ETS1/2 and Mesp homologues of cardiogenic transcription factors of Ciona intestinalis, to convert primary human dermal fibroblasts into cardiac progenitors. Here we show murine Ets2 has an obligatory role for directing cardiac progenitors during cardiopoesis in embryonic stem cells. ETS2 converted fibroblasts into KDR/Flk1+ replicative cells but, like the purported cardiac master regulatory gene Mesp1, could not by itself generate cardiac progenitors de novo from fibroblasts. Co-expression of both Ets2 and Mesp1, however, successfully reprogrammed differentiated fibroblasts into cardiac progenitors, as shown by the de novo appearance of core cardiac transcription factors, gap junction proteins, sarcomeric proteins, electrical activity and contractility. ETS2 and Mesp1 sit at the pinnacle of the cardiopoesis regulatory hierarchy and are well suited for treating human heart disease. Co-expression of both Ets2 and Mesp1, reprogrammed differentiated fibroblasts into cardiac progenitors All sample were done in triplicates, controls were NHDF and ETS2 only infected cells. NHDF were first infected with Doxycyline redulated (Doxy-) ETS2 lentivirus and supplemented with doxycycline for 1 week, sequentially cells were infected with Doxy-Mesp1 and treated for 1 more week. Cells were then aggegated to form EB and hangdrop for 1 week, at the end of that period cells were plated and samples were taken every 24 hrs
Project description:Expression profiling of HeLa cells transduced stably with pre-miR-31 mimic, miR-31 inhibitor and parental HeLa cells was performed using Human Whole Genome OneArray v6.1 (Phalanx Biotech Group, Taiwan, China). Four hybridizations for each group were performed, with two biological and two technical replicates. Briefly, the signal intensity of each spot was loaded into the Rosetta Resolver System (Rosetta Biosoftware, Cambridge, MA, USA) for data analysis. The technical repeat data were tested by Pearson's correlation coefficient calculation to check the reproducibility (R>0.95). Normalized spot intensities were transformed to gene expression log2 ratios between the control and treatment groups.
Project description:Most human transcripts are alternatively spliced, and many disease-causing mutations affect RNA splicing. Towards better modeling the sequence determinants of alternative splicing, we measured the splicing patterns of nearly 2 million (M) synthetic mini-genes, which include degenerate subsequences totaling to nearly 100M bases of variation. The massive size of these training data allowed us to improve upon current models of splicing as well as to gain new mechanistic insights. Our results show that a vast majority of hexamer sequence motifs measurably influence splice site selection when positioned within alternative exons, with multiple motifs acting additively rather than cooperatively. Intriguingly, motifs that enhance (suppress) exon inclusion in alternative 5’ splicing also enhance (suppress) exon inclusion in alternative 3’ or cassette exon splicing, suggesting a universal mechanism for alternative exon recognition. Finally, our empirically trained models are highly predictive of the effects of naturally occurring variants on alternative splicing in vivo. Overall design: HEK293 cells were transfected with two alternatively spliced plasmid libraries. Spliced reads were sequenced to determine isoform counts for each library sequence.
Project description:RNA sequencing of control or Notch1-expressing mouse cells co-cultured with control, Jag1WT, or Jag1Ndr-expressing human cells. Deep sequencing and bioinformatical separation of mouse and human reads reveals transcripts specifically regulated in mouse receptor-expressing cells. Overall design: Mouse C2C12 control and C2C12-FLNotch1, and human HEK-293-Flp-In cells (Hansson et al., 2010): HEK293-Flp control (Flp Ctrl), HEK293-Flp-Jag1WT (Flp Jag1+), HEK293-Flp-Jag1Ndr (Flp Jag1Ndr) were used in this experiment. In one 12-well plate, we seeded 3 wells of mouse C2C12 control cells and 3 wells of C2C12-FLN1 cells, with 3.6x105 cells in 1 mL antibiotic-free medium per well. Cells were allowed to settle for 8 hours. C2C12 control and C2C12-FLN1 cells were transfected with pcDNA5 (1.6 ug/well). All transfections were done using Lipofectamine® 2000 (InvitrogenTM, cat. no. 11668-019) with Opti-MEM® I Reduced Serum Medium (Gibco®, cat. no. 31985-062), according to manufacturer’s instructions. The following day (18 hours post transfection), 3.6x105 cells in 0.5 mL antibiotic-free medium of Flp Ctrl, Flp Jag1+, or Flp Jag1Ndr cells were added. Cells were co-cultured for 6 hours, then lysed in 350 uL per well Buffer RLT (QIAGEN, cat. no. 79216) with 1% 2-Mercaptoethanol (Sigma-Aldrich®, cat. no. M3148) and stored at -80°C until RNA extraction.
Project description:Receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is expressed during embryogenesis and by certain leukemias, but not by tissues of healthy adults. Here we show that the neoplastic cells of many human breast cancers express the ROR1 protein and high-level expression of ROR1 in breast adenocarcinoma was associated with aggressive disease. Total mRNA from Ct-shRNA MDA-MB-231 stable cell line and ROR1-shRNA MDA-MB-231 stable cell line were labeled with Cy5, hybridized to a human Oligo Microarray (Phalanx Human Whole Genome OneArrayTM, Phalanx Biotech) according to the manufacturer’s protocol. Arrays in duplicates were then scanned using GenePix 4000B scanner (Molecular Devices) and the probe intensities were extracted and processed using the GenePix Pro 6.0 software (Molecular Devices). After removing experimental control probes, the intensities of 30968 probes were quantile normalized to have similar distributions across all 4 arrays. The normalized intensity of probes derived from the same gene was collapsed into a single NCBI Entrez gene by the maximal value in each sample (mapping information is obtained from Phalanx’s probe annotation table). In total, 15598 genes were measured at transcription level. Differential expression of genes was quantified using fold enrichment. Gene functional annotation regarding cell proliferation, cell apoptosis, CREB signaling, breast cancer signature was downloaded from MSigDB database. Gene set enrichment analysis was described by Mootha.