Sökning: onr:"swepub:oai:lup.lub.lu.se:b583b313-e458-490d-8e98-15bec4cb4cab" >
Gene editing of CD3...
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
-
- Alattar, AG (författare)
- Lund University,Lunds universitet,Stamceller till röda blodkroppar,Forskargrupper vid Lunds universitet,Transfusionsmedicin,Stem Cells to Red Blood Cells,Lund University Research Groups,Transfusion Medicine
-
- Bäckström, A (författare)
- Lund University,Lunds universitet
-
- Flygare, J (författare)
- Lund University,Lunds universitet,Stamceller till röda blodkroppar,Forskargrupper vid Lunds universitet,Stem Cells to Red Blood Cells,Lund University Research Groups
-
visa fler...
-
- Storry, J (författare)
- Lund University,Lunds universitet,Avdelningen för hematologi och transfusionsmedicin,Institutionen för laboratoriemedicin,Medicinska fakulteten,Transfusionsmedicin,Forskargrupper vid Lunds universitet,Division of Hematology and Transfusion Medicine,Department of Laboratory Medicine,Faculty of Medicine,Transfusion Medicine,Lund University Research Groups,Skåne University Hospital
-
- Larsson, J (författare)
- Lund University,Lunds universitet,WCMM- Wallenberg center för molekylär medicinsk forskning,Medicinska fakulteten,WCMM-Wallenberg Centre for Molecular Medicine,Faculty of Medicine
-
- Olsson, ML (författare)
- Lund University,Lunds universitet,Transfusionsmedicin,Forskargrupper vid Lunds universitet,Transfusion Medicine,Lund University Research Groups,Region Skåne
-
visa färre...
-
(creator_code:org_t)
- 2020-12-10
- 2020
- Engelska.
-
Ingår i: Vox Sanguinis. - : Wiley. - 1423-0410 .- 0042-9007. ; 115:Suppl. s1, s. 363-363
- Relaterad länk:
-
http://dx.doi.org/10... (free)
-
visa fler...
-
https://onlinelibrar...
-
https://lup.lub.lu.s...
-
https://doi.org/10.1...
-
visa färre...
Abstract
Ämnesord
Stäng
- 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
Ämnesord
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Cell- och molekylärbiologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Cell and Molecular Biology (hsv//eng)
Publikations- och innehållstyp
- kon (ämneskategori)
- ref (ämneskategori)
Hitta via bibliotek
Till lärosätets databas