| 1. |
- Bjarnegård, Mattias, 1970, et al.
(författare)
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Endothelium-specific ablation of PDGFB leads to pericyte loss and glomerular, cardiac and placental abnormalities
- 2004
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Ingår i: DEVELOPMENT. - : The Company of Biologists. - 0950-1991 .- 1477-9129. ; 131:8, s. 1847-1857
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Tidskriftsartikel (refereegranskat)abstract
- Platelet-derived growth factor-B (PDGFB) is necessary for normal cardiovascular development, but the relative importance of different cellular sources of PDGFB has not been established. Using Cre-lox techniques, we show here that genetic ablation of Pdgfb in endothelial cells leads to impaired recruitment of pericytes to blood vessels. The endothelium-restricted Pdgfb knockout mutants also developed organ defects including cardiac, placental and renal abnormalities. These defects were similar to those observed in Pdgfb null mice. However, in marked contrast to the embryonic lethality of Pdgfb null mutants, the endothelium-specific mutants survived into adulthood with persistent pathological changes, including brain microhemorrhages, focal astrogliosis, and kidney glomerulus abnormalities. This spectrum of pathological changes is reminiscent of diabetic microangiopathy, suggesting that the endothelium-restricted Pdgfb knockouts may serve as models for some of the pathogenic events of vascular complications to diabetes. Key words: PDGFB, Endothelium, Cre, loxP, Pericytes, Microaneurysm
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| 2. |
- Fredriksson, Simon, et al.
(författare)
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Protein detection using proximity-dependent DNA ligation assays
- 2002
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Ingår i: Nature Biotechnology. - : Springer Science and Business Media LLC. - 1087-0156 .- 1546-1696. ; 20:5, s. 473-477
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Tidskriftsartikel (refereegranskat)abstract
- The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe. The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 x 10(-21) mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.
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| 3. |
- Fredriksson, Simon, 1973-
(författare)
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Proximity Ligation : Transforming protein analysis into nucleic acid detection through proximity-dependent ligation of DNA sequence tagged protein-binders
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A novel technology for protein detection, proximity ligation, has been developed along with improved methods for in situ synthesis of DNA microarrays. Proximity ligation enables a specific and quantitative transformation of proteins present in a sample into nucleic acid sequences. As pairs of so-called proximity probes bind the individual target protein molecules at distinct sites, these reagents are brought in close proximity. The probes consist of a protein specific binding part coupled to an oligonucleotide with either a free 3’- or 5’-end capable of hybridizing to a common connector oligonucleotide. When the probes are in proximity, promoted by target binding, then the DNA strands can be joined by enzymatic ligation. The nucleic acid sequence that is formed can then be amplified and quantitatively detected in a real-time monitored polymerase chain reaction. This convenient assay is simple to perform and allows highly sensitive protein detection. Parallel analysis of multiple proteins by DNA microarray technology is anticipated for proximity ligation and enabled by the information carrying ability of nucleic acids to define the individual proteins. Assays detecting cytokines using SELEX aptamers or antibodies, monoclonal and polyclonal, are presented in the thesis.Microarrays synthesized in situ using photolithographic methods generate impure products due to damaged molecules and interrupted synthesis. Through a molecular inversion mechanism presented here, these impurities may be removed. At the end of synthesis, full-length oligonucleotides receive a functional group that can then be made to react with the solid support forming an arched structure. The 3’-ends of the oligonucleotides are then cleaved, removing the impurities from the support and allowing the liberated 3’-hydroxyl to prime polymerase extension reactions from the inverted oligonucleotides. The effect of having pure oligonucleotides probes compared to ones contaminated with shorter variants was investigated in allele specific hybridization reactions. Pure probes were shown to have greater ability to discriminate between matched and singly mismatched targets at optimal hybridization temperatures.
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| 4. |
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| 5. |
- Gullberg, Mats, et al.
(författare)
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Cytokine detection by antibody-based proximity ligation
- 2004
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 101:22, s. 8420-4
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Efficient and precise detection techniques, along with extensive repertoires of specific binding reagents, will be needed to meet the challenges of proteome analyses. The recently established proximity ligation mechanism enables sensitive high-capacity protein measurements by converting the detection of specific proteins to the analysis of DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins and to form amplifiable tag sequences by ligation when brought in proximity. In our previous report, both the ligatable arms and the protein binders were DNA molecules. We now generalize the method by providing simple and convenient protocols to convert any polyclonal antibodies or matched pair of monoclonal antibodies to proximity probe sets through the attachment of oligonucleotide sequences. Sufficient reagent for >100,000 proximity ligation assays can be prepared from 1 microg of antibody. The technique is applied to measure cytokines in a homogenous test format with femtomolar detection sensitivities in 1-microl samples, and we exemplify its utility in situations when only minute sample amounts are available.
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| 6. |
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| 7. |
- Landegren, Ulf, et al.
(författare)
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Molecular tools for a molecular medicine : analyzing genes, transcripts and proteins using padlock and proximity probes
- 2004
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 17:3, s. 194-7
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Tidskriftsartikel (refereegranskat)abstract
- Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.
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| 8. |
- Landegren, Ulf, et al.
(författare)
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Padlock and proximity probes for in situ and array-based analyses : tools for the post genomic era
- 2003
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Ingår i: Comparative and functional genomics. - : Hindawi Limited. - 1531-6912 .- 1532-6268. ; 4:5, s. 525-30
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Tidskriftsartikel (refereegranskat)abstract
- Highly specific high-throughput assays will be required to take full advantage of the accumulating information about the macromolecular composition of cells and tissues, in order to characterize biological systems in health and disease. We discuss the general problem of detection specificity and present the approach our group has taken, involving the reformatting of analogue biological information to digital reporter segments of genetic information via a series of DNA ligation assays. The assays enable extensive, coordinated analyses of the numbers and locations of genes, transcripts and protein.
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| 9. |
- Lindblom, Per, 1974, et al.
(författare)
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Endothelial PDGF-B retention is required for proper investment of pericytes in the microvessel wall.
- 2003
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Ingår i: Genes & development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 17:15, s. 1835-40
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Tidskriftsartikel (refereegranskat)abstract
- Several platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) family members display C-terminal protein motifs that confer retention of the secreted factors within the pericellular space. To address the role of PDGF-B retention in vivo, we deleted the retention motif by gene targeting in mice. This resulted in defective investment of pericytes in the microvessel wall and delayed formation of the renal glomerulus mesangium. Long-term effects of lack of PDGF-B retention included severe retinal deterioration, glomerulosclerosis, and proteinuria. We conclude that retention of PDGF-B in microvessels is essential for proper recruitment and organization of pericytes and for renal and retinal function in adult mice.
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