SUCCESSFUL TRANSFECTION OF JAK2V617F LENTIVIRUS

Abstract

Objectives: The Janus Kinase 2 gene (JAK2) codes for a tyrosine kinase (JAK2) that is associated with the cytoplasmic portion of a variety of transmembrane cytokine and growth factor receptors important for signal transduction in hematopoietic cells. Signaling via JAK2 activation leads to cell growth and differentiation. BCR-ABL1- negative myeloproliferative neoplasms (MPNs) frequently harbor an acquired single nucleotide mutation in JAK2 characterized as Val617Phe (V617F) in the protein. This mutation is identified overall in approximately two-thirds of all MPNs. Lentiviral vectors are commonly used as gene delivery vehicles. In this study, it is aimed to produceJAK2V617F virus.Methods: E. coli DH5α strain was transformed with lentiviral transfer vectors (LTVs) which have the wild type (wt) JAK2 or JAK2 with V617F mutation insert, which also have GFP (Green Fluorescent Protein) gene. LTV that carry only GFP insert, and two additional plasmids (pCl- VSVG, pCPRDEnv) required for viral packaging were also transferred into the bacteria separately by electroporation. Some colonies of transformed bacteria were selected and produced in LB medium. Plasmid DNAs were isolated from bacterial cells and subjected to restriction digestion to confirm correctness of the plasmids. Next, 293T (Human Embryonic Kidney) endothelial cells were transfected with the isolated plasmid vectors using lipofectamine. Transfection efficiency was evaluated by flow cytometry analysis.Results: The plasmid DNAs (JAK2wt-GFP, JAK2V617F-GFP, GFP, pCl-VSVG, pCPRDEnv) were confirmedby digestion with proper restriction enzymeson the agarose gel electrophoresis. 60% and/or highertransfection efficiency was detected in the each cell linewith JAK2wt-GFP, JAK2V617F-GFP and only GFP by flowcytometry analysis. Viral particles were harvested fromthe supernatant of these cell lines by ultracentrifugation.The green color of GFP proteins were also detected underthe light microscope.Conclusion: Lentiviral vector plasmids were clonedin E. coli cells and lentiviral particles were produced in293T cells successfully. This method provides variety ofopportunities for understanding the effects of JAK2V617Fmutation on individual cell types including hematopoieticcell and stem cell. In conclusion, our study could providean understanding of the JAK2V617F mutation effects onthe cells in the context of MPNs.