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- Hilson, P., et al.
(författare)
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Versatile gene-specific sequence tags for Arabidopsis functional genomics : Trancript profiling and reverse genetics applications
- 2004
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Ingår i: Genome Research. - : Cold Spring Harbor Laboratory. - 1088-9051 .- 1549-5469. ; 14:10B, s. 2176-2189
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Tidskriftsartikel (refereegranskat)abstract
- Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.
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| 5. |
- Andersson, T., et al.
(författare)
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Novel candidate genes for atherosclerosis are identified by representational difference analysis-based transcript profiling of cholesterol-loaded macrophages
- 2001
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Ingår i: Pathobiology (Basel). - : S. Karger AG. - 1015-2008 .- 1423-0291. ; 69:6, s. 304-314
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: To analyze the early gene expression in macrophages accompanying the phenotypic changes into foam cells upon exposure to oxidized low-density lipoprotein. To identify candidate genes and markers for further studies into the pathogenesis of atherosclerosis. Methods: Cells of the monocytic cell line THP-1 were activated by PMA and exposed to oxidized low-density lipoprotein. Gene expression profiles were investigated after 24 h, using a solid phase cDNA representational difference analysis (RDA) method and shotgun sequencing. Results were verified by microarray hybridization, and analyzed in the virtual chip display of a novel software tool for transcript profile exploration. Results: By comparing transcript profiles of exposed/unexposed cells, 1,984 transcript sequences, representing a total of 921 genes with altered expression levels in response to oxidized low-density lipoprotein exposure, were identified. Genes that are central to cell cycle control and proliferation, inflammatory response, and of pathways not previously implicated in atherosclerosis were identified. The data obtained is also made available on-line at http:// biobase.biotech.kth.se/thp1a for further exploration. Conclusion: The identification of new candidate genes for atherosclerotic disease through RDA-based transcript profiling facilitates further functional genomic studies in coronary artery disease. Candidate genetic polymorphism markers of potential clinical relevance can be identified by filtering information in genome variation databases through the virtual chip analysis of the transcript profiles and subsequently tested in association studies.
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| 7. |
- Andersson, T., et al.
(författare)
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Shotgun sequencing and microarray analysis of RDA transcripts
- 2003
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Ingår i: Gene. - : Elsevier BV. - 0378-1119 .- 1879-0038. ; 310, s. 39-47
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Tidskriftsartikel (refereegranskat)abstract
- Monitoring of differential gene expression is an important step towards understanding of gene function. We describe a comparison of the representational difference analysis (RDA) subtraction process with corresponding microarray analysis. The subtraction steps are followed in a quantitative manner using a shotgun cloning and sequencing procedure that includes over 1900 gene sequences. In parallel, the enriched transcripts are spotted onto microarrays facilitating large scale hybridization analysis of the representations and the difference products. We show by the shotgun procedure that there is a high diversity of gene fragments represented in the iterative RDA products (92-67% singletons) with a low number of shared sequences (<9%) between subsequent subtraction cycles. A non redundant set of 1141 RDA clones were immobilized on glass slides and the majority of these clones (97%) gave repeated good fluorescent signals in a subsequent hybridization of the labelled and amplified original cDNA. We observed only a low number of false positives (<2%) and a more than twofold differential expression for 32% (363) of the immobilized RDA clones. In conclusion, we show that by random sequencing of the difference products we obtained an accurate transcript profile of the individual steps and that large-scale confirmation of the obtained transcripts can be achieved by microarray analysis.
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| 9. |
- Geale, Kirk, et al.
(författare)
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Persistence of biologic treatment in psoriatic arthritis : a population-based study in Sweden
- 2020
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ Publishing Group Ltd. - 0003-4967 .- 1468-2060. ; 79, s. 37-38
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Psoriatic arthritis (PsA) is a chronic, heterogeneous, immune-mediated seronegative arthritis characterized by joint inflammation in people with skin psoriasis (PsO). In recent years several effective biologic treatments such as tumour necrosis factor inhibitors (TNFi), interleukin (IL) 12 and 23 inhibitors (IL-12/23i), and IL 17 inhibitors (IL-17i) have been introduced for PsA. Discontinuation (non-persistence) of therapy is usually a consequence of lack of effect and intolerability.Objectives:Compare time to discontinuation of TNFi (adalimumab, ADA), IL-17i (secukinumab, SEC), and IL-12/23i (ustekinumab, UST) treatment exposures and the association with previous biologic treatment experience.Methods:Population-based national health data from the Swedish Patient Registry, Prescribed Drug Registry and Cause of Death Registry were linked at the patient level and used to identify treatment exposures in PsA patients initiating ADA, SEC, or UST between January 2008 and September 2018. Discontinuation was defined as a treatment switch to any other PsA-indicated biologic, or failure to re-dispense treatment within a grace period following end of drug supplied. The grace period, defined as the number of days between end of drug supply and re-dispensation during which a patient is considered to be on active treatment, was set dynamically to the number of days of drug supplied in the primary analysis. As a sensitivity analysis, a fixed 90-day grace period was used. Supply was calculated as total milligrams dispensed divided by maintenance dose posology, where the following assumptions were made due to the limitations of the administrative data used: UST patients’ weight corresponded to the amount of drug dispensed (both 45mg and 90mg dispensations last 84 days), SEC patients with prior TNFi experience consumed 300mg/28 days and all others consumed 150mg/28 days, and ADA patients consumed 40mg/14 days. Adjusted hazard ratios (HR) for time to discontinuation were calculated using a Cox proportional hazards model. Covariates for age, marital status, and previous biologic treatment experience were assessed at the initiation of treatment exposure, while comorbidity including skin PsO was assessed during the two years prior. Exposures without discontinuation events were censored at death or end of follow-up. The study was approved by the Stockholm Regional Ethical Review Board.Results:3,620 discontinuation events were observed in the main analysis across 4,649 treatment exposures (ADA: 3,255; SEC: 887; UST: 507) (Figure 1, unadjusted). 3,162 events were observed in the sensitivity analysis. Average age at treatment initiation was 50, 54% were female, 47% were biologic treatment naïve, and 39% had skin PsO. In the multivariate main analysis, UST exhibited lower discontinuation rates vs ADA (HR=0.56, 95% CI: 0.49-0.64) while there was no significant difference between SEC and ADA (HR=1.01, 95% CI: 0.88-1.15). In the multivariate sensitivity analysis, both UST (HR=0.81, 95% CI: 0.70-0.94) and SEC (HR=0.82, 95% CI: 0.70-0.95) were associated with significantly lower discontinuation rates ratio relative to ADA. Overall, patients with more biologic treatment experience were statistically significantly (p<0.05) associated with higher risk of treatment discontinuation.Figure 1.Unadjusted Kaplan-Meier curves of time to treatment discontinuation (main analysis, dynamic grace period)Conclusion:UST exhibits a favourable treatment persistency profile relative to ADA, regardless of the grace period definition. The relative risk of discontinuing SEC vs ADA is sensitive to the grace period. Treatment discontinuation was higher in treatment exposures with more biologic experience.Disclosure of Interests:Kirk Geale Consultant of: Quantify Research, Speakers bureau: Indirectly as a consultant, Ingrid Lindberg Consultant of: Quantify Research, Emma Paulsson Consultant of: Quantify Research, Christina Wennerström Employee of: Janssen-Cilag Sweden AB, Anna Tjärnlund Employee of: Janssen-Cilag Sweden AB, Virginia Taliadouros Shareholder of: JnJ, Employee of: Janssen Pharmaceuticals NV, Wim Noel Employee of: Janssen Pharmaceuticals NV, Dana Enkusson Employee of: Janssen-Cilag AB, Elke Theander Employee of: Janssen-Cilag Sweden AB, Sara Bruce Wirta Employee of: Janssen-Cilag Sweden AB
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