Construction of retrovirus vector of IK4 gene mutation and its expression in leukemic cells.

IK4 is an isoform of IKAROS gene which plays a key role in the hematopoiesis, in this work, the construction of retro-virus vector of IK4 mutation and its expression in leukemic cells was investigated. PCR site-directed mutagenesis method based on overlap extension was used to construct four IK4 gene mutants of single- or multi-site mutations, which were then cloned into the retrovirus vector Pmscv-puro to form recombinant expression vector. The point mutations were verified by DNA sequencing. The recombinant expression vectors were co-transfected with packaging plasmids VSVG and Gag-Pol into 293T cells to produce retroviruses. Supernatants containing retroviruses were collected and were used to infect NB4 cells. Puromycin was used to select stable cell lines. The expression of mutants was verified by Western blotting analysis. Four IK4 gene mutants of single- or multi-site mutations were successfully constructed by overlap extension PCR, and the mutants were expressed in leukemic cells, which will provide basis for subsequent function and mechanism research. [ABSTRACT FROM AUTHOR]