title: Transient expression assay of Agamma-588 (A/G) mutations in the K562 cell line.
abstract: BACKGROUND
  In the previous study, we have shown that the presence of A allele at position -588 in Agamma-globin gene was highly frequent and closely associated with fetal hemoglobin elevation among beta-thalassemia intermedia patients. Therefore, we decided to investigate whether this allele (A allele at -588) could result in an increase in Agamma-globin gene expression to ameliorate the severity of the disease in thalassemia patients.
  
  
  METHODS
  Three constructs containing mu locus control region, Agamma-globin and beta-globin genes were designed and employed in the transient expression assay. The difference among constructs was in the promoter region of Agamma-globin gene (A and G alleles at -588). A construct with T to C base substitution at -175 of Agamma-globin, created by site-directed mutagenesis, was selected as positive control. The K562 cell line was transfected with the above constructs. Subsequently, the expression of Agamma-globin gene was determined by quantitative real-time reverse transcription-PCR.
  
  
  RESULTS
  There was not a significant increase in the expression of Agamma-globin gene in the construct containing A allele comparing the one with G allele at -588.
  
  
  CONCLUSIONS
  -588 (A>G) mutation does not play a major role in regulation of Agamma-globin gene, suggesting that other factors may be involved.
authors: Molecular Medicine Division, Biotechnology Research Center, Pasteur Institute of Iran, Tehran.
authors: Mohammad
authors: M
authors: Hamid
authors: Frouzandeh
authors: F
authors: Mahjoubi
authors: Mohammad Taghi
authors: MT
authors: Akbari
authors: Hossein
authors: H
authors: Khanahmad
authors: Fatemeh
authors: F
authors: Jamshidi
authors: Sirous
authors: S
authors: Zeinali
authors: Morteza
authors: M
authors: Karimipoor
mesh_terms: D018274:Electroporation; D005434:Flow Cytometry; D015870:Gene Expression; D015973:Gene Expression Regulation, Leukemic; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D009154:Mutation / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D055545:gamma-Globins / Q000235:genetics* / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D055545:gamma-Globins; D049452:Green Fluorescent Proteins