title: [Construction of AML1-ETO eukaryotic expression vector and its effects on proliferation and differentiation of U937 cells].
abstract: OBJECTIVE
  To construct a pcDNA3.1-AML1-ETO expression vector and investigate its effects on proliferation and differentiation of U937 leukemic cells.
  
  
  METHODS
  AML1-ETO gene was amplified by PCR from pCMV5-AML1-ETO and inserted into eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO. The recombinant plasmid was transfected into U937 cells by Lipofectamin 2000. Individual clones selected with G418 were isolated. The integration and the expression levels of AML1-ETO in transfectants were determined by PCR, RT-PCR and Western blot analysis respectively. Trypan blue refusal staining method was used to detect the proliferation of U937 cells. Light microscope was applied to observe the morphologic changes of the cell. The expression of myeloid cell differentiation antigen was detected using flow cytometry.
  
  
  RESULTS
  The recombinant pcDNA3.1-AML1-ETO was confirmed by enzyme digestion and sequencing. The highly expressing AML1-ETO subclone was established. AML1-ETO was expressed in U937 cells transfected with pcDNA3.1-AML1-ETO. The growth of the monoclonal cells was inhibited evidently (P < 0.05). The expression of CD11b in transfected group \[(4.17 ± 0.31)%\] was lower than that in empty plasmid transfected group and non-transfected group \[(11.40 ± 0.17)% and (11.03 ± 0.15)%\] respectively (P < 0.001). Transfected cells displayed morphology of less differentiation. The expression level of CDl1b was unchanged in transfected cells treated with TPA (P > 0.05).
  
  
  CONCLUSIONS
  The eukaryotic expression vector for AML1-ETO gene was successfully constructed and expressed in U937. AML1-ETO inhibits the proliferation and differentiation of transfected cells. It provides the basis for further study of mechanisms of AML1-ETO in leukemogenesis.
authors.affiliation: Medical College of Soochow University, the First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Suzhou 215000, China.
authors.forename: Wen-Yue
authors.initials: WY
authors.lastname: Zhuang
authors.forename: Zi-Xing
authors.initials: ZX
authors.lastname: Chen
authors.forename: Xiao-Fei
authors.initials: XF
authors.lastname: Qi
authors.forename: Jian-Nong
authors.initials: JN
authors.lastname: Cen
authors.forename: Hong-Jie
authors.initials: HJ
authors.lastname: Shen
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D002454:Cell Differentiation; D049109:Cell Proliferation; D050676:Core Binding Factor Alpha 2 Subunit / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007938:Leukemia / Q000235:genetics / Q000473:pathology*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D010957:Plasmids; D000075142:RUNX1 Translocation Partner 1 Protein; D020298:U937 Cells
chemical_list: C107844:AML1-ETO fusion protein, human; D050676:Core Binding Factor Alpha 2 Subunit; D015514:Oncogene Proteins, Fusion; D000075142:RUNX1 Translocation Partner 1 Protein