Methods for genetic modification of megakaryocytes and platelets

Pendaries, Caroline; Watson, Steve P.; Spalton, Jennifer C.

doi:10.1080/09537100701288012

Cited by 7 publications

(3 citation statements)

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“…MKs are PLT precursor cells, contain many of the same proteins and pathways as PLTs, and are often used as a relevant model system to study PLT responses [ 8 , 60 , 61 ]. While PLTs are only extremely difficult or not at all accessible to modifications of their transcriptome, MKs have been genetically modified using several different techniques [ 62 , 63 ]. Here, we used “liposome-based transfection” utilizing the transfer of in vitro transcribed, capped, and poly(A) tailed NAP1L1 mRNA into MKs ( Figure 7 A), to induce transient overexpression of NAP1L1.…”

Section: Resultsmentioning

confidence: 99%

A New Role of NAP1L1 in Megakaryocytes and Human Platelets

Freitag

Schwertz

2022

IJMS

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Platelets (PLTs) are anucleate and considered incapable of nuclear functions. Contrastingly, nuclear proteins were detected in human PLTs. For most of these proteins, it is unclear if nuclear or alternatively assigned functions are performed, a question we wanted to address for nuclear assembly protein 1like 1 (NAP1L1). Using a wide array of molecular methods, including RNAseq, co-IP, overexpression and functional assays, we explored expression pattern and functionality of NAP1L1 in PLTs, and CD34+-derived megakaryocytes (MKs). NAP1L1 is expressed in PLTs and MKs. Co-IP experiments revealed that dihydrolipolylysine-residue acetyltransferase (DLAT encoded protein PDC-E2, ODP2) dynamically interacts with NAP1L1. PDC-E2 is part of the mitochondrial pyruvate-dehydrogenase (PDH) multi-enzyme complex, playing a crucial role in maintaining cellular respiration, and promoting ATP-synthesis via the respiratory chain. Since altered mitochondrial function is a hallmark of infectious syndromes, we analyzed PDH activity in PLTs from septic patients demonstrating increased activity, paralleling NAP1L1 expression levels. MKs PDH activity decreased following an LPS-challenge. Furthermore, overexpression of NAP1L1 significantly altered the ability of MKs to form proplatelet extensions, diminishing thrombopoiesis. These results indicate that NAP1L1 performs in other than nucleosome-assembly functions in PTLs and MKs, binding a key mitochondrial protein as a potential chaperone, and gatekeeper, influencing PDH activity and thrombopoiesis.

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Section: Resultsmentioning

confidence: 99%

A New Role of NAP1L1 in Megakaryocytes and Human Platelets

Freitag

Schwertz

2022

IJMS

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“…We speculated that these cell type–specific results, which was previously addressed by Pendaries et al . , are partially due to cell type–specific interacting proteins. To overcome this, we had recently established an ES cell differentiation system in which exogenous RUNX1 could be overexpressed in a megakaryocytic lineage‐specific manner during megakaryocytic differentiation .…”

Section: Discussionmentioning

confidence: 99%

RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4gene expression in combination with ETS family proteins

Okada

Watanabe

Nakai

et al. 2013

Journal of Thrombosis and Haemostasis

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WC, Doi T. RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4 gene expression in combination with ETS family proteins. J Thromb Haemost 2013; 11: 1742-50.Summary. Background: Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runtrelated transcription factor 1 (RUNX1) of FPD patients. Objectives: To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1-mediated induction of the FPD phenotype. Methods: We assessed whether RUNX1 and its mutants, in combination with E26 transformation-specific-1 (ETS-1), Core-binding factor subunit beta (CBFb), and Friend leukemia virus integration 1 (FLI-1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real-time RT-PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos-7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos-7 cells. Results: We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS-1 and CBFb, or FLI-1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA-binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS-1, CBFb, and FLI-1. Conclusions: RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.

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“…1a). Platelets play a critical role in homeostasis: patients with a deficient number or abnormality of platelets often experience excessive bleeding (Pendaries et al 2007), which could lead to a life-threatening condition.…”

Section: Introductionmentioning

confidence: 99%

Recent developments in ex vivo platelet production

2016

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The platelet is a component of blood that functions to initiate blood clotting. Abnormal platelet count and function can lead to a life-threatening condition caused by excessive bleeding. At present, platelet supply for transfusion can be obtained only from platelet donation. However, platelets cannot be stored for longer than 7 days, meaning that routine isolation is required to maintain platelet supply for transfusion. To mitigate for potential platelet shortages, several strategies have been proposed to generate platelets ex vivo. By employing both of natural and artificial approaches, several researchers have successfully generated biomaterials with characteristics similar to human-derived platelets. Their reports indicated that the biomaterials could mimic the aggregation of human-isolated platelets, further suggesting the possibility to substitute or complement human-isolated platelets. The current review summarizes the progress in ex vivo platelet production and gives a prospect for the possible approaches to achieving a feasible platelet factory, based on the Good Manufacturing Practice standards.

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Methods for genetic modification of megakaryocytes and platelets

Cited by 7 publications

References 100 publications

A New Role of NAP1L1 in Megakaryocytes and Human Platelets

A New Role of NAP1L1 in Megakaryocytes and Human Platelets

RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4gene expression in combination with ETS family proteins

Recent developments in ex vivo platelet production

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