Akademik Veri Yönetim Sistemi
5. International Congress on Leukemia Lymphoma and Myeloma, İstanbul, Türkiye, 21 - 24 Mayıs 2015, ss.148-149
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 produce
JAK2V617F 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 confirmed
by digestion with proper restriction enzymes
on the agarose gel electrophoresis. 60% and/or higher
transfection efficiency was detected in the each cell line
with JAK2wt-GFP, JAK2V617F-GFP and only GFP by flow
cytometry analysis. Viral particles were harvested from
the supernatant of these cell lines by ultracentrifugation.
The green color of GFP proteins were also detected under
the light microscope.
Conclusion: Lentiviral vector plasmids were cloned
in E. coli cells and lentiviral particles were produced in
293T cells successfully. This method provides variety of
opportunities for understanding the effects of JAK2V617F
mutation on individual cell types including hematopoietic
cell and stem cell. In conclusion, our study could provide
an understanding of the JAK2V617F mutation effects on
the cells in the context of MPNs.