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- Alattar, AG, et al.
(författare)
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Gene editing of CD34+ progenitor cells from single blood donor waste bags to create cultured early erythroid cells for study of blood group knock-outs
- 2020
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Ingår i: Vox Sanguinis. - : Wiley. - 1423-0410 .- 0042-9007. ; 115:Suppl. s1, s. 363-363
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Konferensbidrag (refereegranskat)abstract
- Background: Many blood group antigens are carried by red cell surface proteins, thefunctions of which are not yet fully characterized or in some cases completelyunknown. By investigating red cells with naturally-occurring blood group variants,much has been learnt about the underlying molecules. However, interindividualvariation affecting other molecules than the one(s) of interest may confound orotherwise hamper such studies. As an alternative, a reductionistic approach whereonly a single factor differs between test and control cells significantly facilitatesinterpretation of functional studies. Using various siRNA, shRNA and various gene-editing tools blood group expression can be manipulated and useful modelsdeveloped. Applying the latter on primary hematopoietic stem and progenitor cells(HSPCs) can be challenging.Aims: We evaluated a protocol for gene editing of CD44 using a CRISPR/Cas9hybrid system on HSPCs isolated from blood donation leukocyte waste bags fromsingle donors to develop a model for study of blood group molecular function inerythropoiesis.Methods: Peripheral blood mononuclear cells (PBMCs) were from anonymizedleucocyte waste bags obtained after whole blood unit processing in the Reveosautomated blood component system. Cells were harvested following Lymphoprepgradient separation and CD34+HSPCs enriched and collected using magnetic beads.CD34+cells were cultured in 2-phase culture medium to generate erythroid cellsfrom HSPCs (Vidovic, Vox Sang 2017). For CRISPR/Cas9 gene editing, a short guideRNA (sgRNA) targeting CD44 was designed and cloned into the lentiCRISPR v2vector (Addgene plasmid #52961). A non-targeting sgRNA cloned into the vectorwas used as control. Lentivirus particles were produced in the human 293T cell lineas described previously (Galeev, Methods Mol Biol 2017). Equal number of CD34+cells were transduced 24 hours after collection using RetroNectin following theRetroNectin-Bound Virus (RBV) Infection Method according to manufacturer’sprotocol. Cells were transduced at a multiplicity of infection (MOI) of 10 with atarget transduction efficiency of 20–30%. Cells were cultured at 37°C, 5% CO2 for72 hours in phase I culture medium and then electroporated with Cas9 mRNA usingthe ECM 830 Electroporation System as described previously for cord blood-derivedCD34+cells (Backstrom, Exp Hematol 2019). GFP+CD44-edited cell frequencieswere monitored by flow cytometry at day 7 of the HSPC expansion phase and atday 14 of the erythroid expansion-differentiation phase using antibodies againsterythroid-specific cell surface markers GPA and Band3 in addition to CD49d toassess the erythroid development stage.Results: We tested the above protocol and observed that the frequencies of editedcells lacking CD44 expression within the GFP+population at day 7 of culture were10–25% while the edited frequencies were increased at day 14 of culture to 60–80%within the GFP+cells. Whilst this stage corresponds to erythroblasts, earlier or laterstages can be tested.Summary/Conclusions: The previously established hybrid system for CRISPR/Cas9gene editing in cord blood-derived CD34+HSPCs, which combines lentiviraldelivery of the sgRNA with transient delivery of Cas9 mRNA by electroporation, isalso applicable to primary human adult HSPCs from Reveos-processed single-donorwhole blood donation, resulting in a traceable high-yield gene-editing system tostudy blood group function and erythroid development
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- Dahlbom, I, et al.
(författare)
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Immunoglobulin g (IgG) anti-tissue transglutaminase antibodies used as markers for IgA-deficient celiac disease patients
- 2005
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Ingår i: Clinical and Diagnostic Laboratory Immunology. - 1071-412X. ; 12:2, s. 254-258
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Tidskriftsartikel (refereegranskat)abstract
- The role of immunoglobulin A (IgA) anti-tissue transglutaminase antibodies (IgA-tTG) as predictors of untreated celiac disease (CoD) is well documented, and the presence and levels of these antibodies are most accurately monitored with native or recombinant human antigens. However, IgA-deficient CoD patients are not identified by IgA serology, and conflicting results concerning the diagnostic validity of IgG antibodies against gliadin (IgG-AGA), endomysium (IgG-EmA), and tTG (IgG-tTG) have been reported. The aim of the present study was to evaluate the utility of IgG-tTG for the detection of CoD in IgA-deficient patients. Samples from 115 IgA-deficient and 200 IgA-sufficient subjects were collected and tested for the presence of IgA and IgG antibodies against tTG, EmA, and AGA. Antibodies against tTG were measured by an enzyme-linked immunosorbent assay based on recombinant human tTG, and antibodies against EmA were determined by immunofluorescence. The values for IgG-tTG showed a higher correlation (correlation coefficient [r] = 0.91) with those for IgG-EmA for the IgA-deficient subjects than for the IgA-sufficient subjects (r = 0.88). The overall concordance of the positive and negative results between IgG-tTG and IgG-EmA was 97%, and the IgG-tTG assay discriminated between IgG-EmA-positive and -negative subjects with IgA deficiency at a rate of 100%. Elevated levels of IgG-tTG and IgG-EmA were measured in 70% of the IgA-sufficient subjects. IgG-tTG detection with recombinant human tTG is a good alternative to IgG-EmA detection, and the addition of IgG-tTG assessment to present screening methods may improve the ability to identify IgA-deficient subjects with CoD.
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- Duffy, DL, et al.
(författare)
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Publisher Correction: Novel pleiotropic risk loci for melanoma and nevus density implicate multiple biological pathways
- 2019
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10:1, s. 299-
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Tidskriftsartikel (refereegranskat)abstract
- The original version of this Article contained errors in the spelling of the authors Fan Liu and M. Arfan Ikram, which were incorrectly given as Fan Lui and Arfan M. Ikram. In addition, the original version of this Article also contained errors in the author affiliations which are detailed in the associated Publisher Correction.
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