Establishment Of Stable Subline Of K562 Cells Overexpressing HMGB1 Protein.

Objective: This study was amided to establish a stable subline of K562 cells (K562-HMGB1) overexpressing HMGB1 protein and K562-pcDNA3.1 cells served as control, to provide a basis for exploring the role of hmgb1 gene in the mechanism of leukemogenesis.Method: Hmgb1 gene was amplified by PCR with its cDNA, which was synthesized by reverse transcription PCR from total RNA extracted from U937 cells. The PCR-amplified hmgb1 gene was ligated into PMD18-T vector (PMD18-T-HMGB1 vector), and then transformed into E.coli strain DH5α. DH5αcontaining PMD18-T-HMGB1 vector were grown overnight on LB agar plate supplemented with 100μg/ml ampicillin. The single ampicillin-selected DH5αclone was picked for culturing 12-16h and then for plasmid extraction. The extracted plasmid was identified to contain hmgb1 gene by double restriction enzymes of KpnI/XhoI. The correctness of hmgb1 sequence was confirmed with DNA sequencing, and the insert of hmgb1 gene contained in PMD18-T-HMGB1 vector was digested with double restriction enzymes of KpnI/XhoI and then ligated into eukaryotic expression vector pcDNA3.1 to form pcDNA3.1-HMGB1 vector. 10μg of pcDNA3.1-HMGB1 or pcDNA3.1 plasmid was separately electroporated into K562 cells. After 48 hours elctroporation the cells were cultured with G418 at a final concentration of 800μg/ml for over 2 months. Finally stably transfected sublines of K562 cells containing hmgb1gene (K562-HMGB1), and of K562 containing pcDNA3.1 vector (K562-pcDNA3.1) served as a control were obtained. The transcriptional or translational expression of hmgb1 gene was detected with RT-PCR and Western Blot respectively, to tesify transfected efficiency and validity of stable sublines of K562-HMGB1. Results: The eukaryotic expression vector pcDNA3.1-HMGB1 plasmid was successfully constructed and was electroporated into K562 cells. The stable subline of K562 cells containing hmgb1 gene overexpressed HMGB1 at the transcriptional or translational level, suggested that stable sublines of K562-HMGB1 cells was successfully established.Conclusion: It is concluded that the stable sublines of K562-HMGB1 cells or K562-pcDNA3.1 cells are successfully established, which provides a basis for exploring the roles and mechanisms of hmgb1 gene in leukemogenesis and development of leukemia.