Abstract:

Objective   To detect mutation in Sin3-interacting domain(SID) and basic helix-loop-helix leucine zipper domain(bHLHzip) of the Mxi1 gene in the leukemia cell line KG-1, and construct a eukaryotic expression vector of Mxi1, and observe expression of the Mxi1 gene after being transfected into eukaryotic cells. Methods    Expression and mutation of the Mxi1 gene in the leukemia cell line KG-1 were analyzed by reverse transcription-polymerase chain reaction(RT-PCR), single strand conformational po-lymorphism (SSCP) and DNA sequencing. The full length cDNA of the Mxi1 gene was inserted into the plasmid pDs-red2-N1 obtained from fetal lymphocytes and KG-1 cells，which was named pDs-red2-N1/Mxi1(wild /mutation type). Then the vector was transfected into COS-7 cells via liposomes. The Mxi1 mRNA expression was detected by RT-PCR and Mxi1 protein expression was detected by flow cytometry and a fluorescence microscope in COS-7 cells after transfection. Results   DNA sequencing showed the 82th code CAT/TAT(His/Tyr) missense mutation in Helix II region in the KG-1 cell line. After the pDs-red2-N1/Mxi1 eukaryotic expressionvector was digested by endonuclease，two fragments of 4700bp and 1845bp in electrophoresis gel were detected. Expression of the Mxi1 gene could be found in COS-7 cells . The positive cell rate reached about 18.07% after transfection. Mxi1 expression lasted about 6 days in COS-7 cells and the peak occurred on  the third day. Conclusion   Mxi1 mutation occurs in the highly conserved domain bHLHzip in the KG-1 cell line. The Mxi1 gene eukaryotic expression vector was successfully constructed and transfected into COS-7 cells.

Key words:  Genes, mxi1; Genes, myc； Vector construction; Gene transfectionMyc