The effect of exogenous in vitro expression of wild type and mutant JAK2 in erythroblast progenitors
Red blood cell (RBC) transfusion has become a routine and indispensable procedure for many clinical purposes. As there is no appropriate alternative, the in vitro manufacture of RBCs is a potential means to ensure an adequate and safe supply of blood products. The aim of this research is to study the effect of exogenous JAK2 (mutant and wild-type) on cultured haematopoietic cells. The main goal is to understand the expression signatures that are different when comparing CD34+ cells that overexpress wild-type Jak2 and CD34+ cells overexpressing mutant Jak2 which is associated with polycythaemia vera disease.
The human JAK2 coding sequence was amplified by PCR and inserted into a linearised pIRES2 vector to create the JAK2-pIRES2 construct. Another two constructs were obtained, namely wtJAK2-pcDNA3 and mutJAK2-pcDNA3 constructs. Transfection experiments and western blot experiments were performed to assess the expression of the Jak2 protein by the constructs. These experiments showed that the JAK2-IRES-GFP transcript was successfully produced as the GFP protein was expressed by the transfected cells. Western blot analysis confirmed the presence of Jak2 through the production of protein bands at 125kDa.
Transient transfections were performed in which CD34+ cells harvested from human buffy coats were transfected with the constructs wtJAK2-pcDNA3 and mutJAK2-pcDNA3 using the Amaxa® Nucleofection protocol. Transfections were performed to overexpress Jak2 and study its effect on CD34+ cells. The expression of Jak2 was confirmed by flow cytometry through the detection of JAK2 mRNA with the target probe. The expansion of cells that were transfected by the wild-type and mutant JAK2 constructs was similar to control cells, suggesting that overexpression of Jak2 in CD34+ cells is not enough to drive over-expansion of cells and perhaps JAK2 does not impact haematopoietic stem cells but has an effect on cells at a different maturity stage. However, transient transfection of mutant JAK2 into CD34+ cells enhanced survival of cells during erythroid selection.
RNA sequencing analysis compared mutJAK2-transfected CD34+ cells to wtJAK2-transfected CD34+ cells providing a differential gene expression profile. The top differentially expressed genes encode ribosomal proteins and translation factors indicating that CD34+ cells that were transfected with mutJAK2 were being prepared for proliferation. Through Ingenuity Pathway Analysis it was observed that NPM1 was overexpressed, that PI3K/PKB/mTOR pathway was activated and that the activity of the upstream regulators MYC, erythropoietin, kit ligand and dexamethasone was enhanced, concluding that mutJAK2 expression prepares the cells for proliferation, differentiation and survival.
This research is the first to study the differential gene expression of mutant Jak2 against wild-type Jak2 expressing cells and is the first study to show an association between the JAK2-V617F mutation and expression of wild-type NPM1 in the context of driving over-expansion of haematopoietic cells.
Aquilina, S. The effect of exogenous in vitro expression of wild type and mutant JAK2 in erythroblast progenitors. (Thesis). University of the West of England
|Keywords||exogenous, blood, JAK2|
Final approved version of thesis - Sephora Aquilina.pdf