Abstract
Objective: Chemokine receptors and their ligands play important roles in the leukemia-initiating cell activity ,BCP-ALL progression and CNS infiltrating. To study the role of chemokine receptor CXCR3 in the mechanism of leukemia cell infiltration of the CNS, CXCR3 overexpression cell lines should be established. Lentivirus generally does not interfere with proto-oncogene and can express long-term and stable, so it is a good vector for genetic research on hematopoietic cells and T cells.
Methods: Mouse CXCR3 gene fragment was extracted by PCR from T vector which was kept in our lab. After the extraction, CXCR3 fragment and linearized LV5 lentivirus overexpressing vector(with GFP+-puro) were digested with NotI and BamHI.After this,we used T4 DNA ligase to connect them, making the target gene (CXCR3) cloned into LV5 vector, to obtain recombinant shuttle plasmid. Next,we mixed the shuttle plasmid with packaging plasmids (pGag/Pol, pRev, pVSV-G), packaged lentivirus, collected, tested viral titer and sequenced target gene by DNA sequencing. Then the constructed CXCR3 overexpression lentivirus transfected Jurkat and L1210 cell lines, at the same time MOI achieved to 100, twice infection and adding polybrene 5ug/ml to increase lentivirus transfection efficiency. After GFP expression was observed under fluorescent microscope, we used puromycin to select transfected cells and tested the expression of CXCR3 on cell surface by flow cytometry analysis of Anti-Mouse CD183 (CXCR3).
Results: After we sequenced the target mouse CXCR3 gene, viral titer reached to 1×10^9 TU/ml and GFP expression was observed after transfection 96h (<10%) on lymphocytic leukemia cell lines. Through puromycin selection, GFP+ cells surpassed 90% and CXCR3 overexpression group had upregulated 30%-90% compared with control group.
Conclusion: Chemokine receptor CXCR3 had been successful transfected into lymphocytic leukemia cell lines and had stably high expression. The effect of CXCR3 in leukemic invasion and leukemia progression can be further studied using constructed CXCR3 overexpression lymphocytic cell lines.
Disclosures
No relevant conflicts of interest to declare.
Tetramethylpyrazine inhibits the proliferation of acute lymphocytic leukemia cell lines via decrease in GSK-3β