| 1. |
- 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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| 2. |
- Åkesson, A, et al.
(författare)
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Characterization of specific IgE response in vitro against protein and drug allergens using atopic and normal donors
- 2002
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Ingår i: Allergy. - : Wiley. - 1398-9995 .- 0105-4538. ; 57:3, s. 193-200
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Tidskriftsartikel (refereegranskat)abstract
- Background: As the incidence of allergy to different compounds increases in society, the need to understand and characterize specific IgE responses becomes F. obvious. Different cell culture systems have been evaluated for their ability to support such IgE secretion. Methods: One system employed human peripheral lymphocytes (PBL) from normal donors stimulated with anti-CD3 activated T cells with or without the presence of allergens like benzylpenicillin (BP) and Phlenum pratense (PP). Secretion of IgE was analyzed in ELISA and compared to the IgG response to the nonallergenic antigen tetanus toxoid (TT). Another system employed stimulation of T and B cells with a heterotope, consisting of a T helper cell epitope derived from TT, and a B cell allergen epitope derived from BP. The specific IgE secretion was compared, using lymphocytes from nor-Mal as well as BP-allergic donors. Results: Anti-CD3 stimulated T cells supported BP-specific IgE secretion in Six of 11 normal donors. This response was inhibited in four donors and enhanced in two donors by the addition of the BP-allergen to the culture. In contrast, addition of the protein allergen (PP) or antigen (TT) to the same culture system inhibited both IgE and IgG synthesis in all experiments. C-ells from the majority (10/16) of the BP-allergic donors failed to produce BP-specific IgE in vitro, when cultured in the presence of allergen. Conclusions: An allergen specific immune response is readily generated in vitro. The differential response against benzylpenicillin between different donor categories most probably reflects the level of pre-exposure to this allergen in vivo.
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