关键词: PCR, 鸡α-珠蛋白基因5′端MAR, pGFP/2MAR, GFP, 流式细胞仪

Abstract: Objective Recently,it has reported that gene expression and regulation in eukaryotic cells were closely related to matrix attachment regions(MARs).MARs,also known as scaffold/matrix attachment regions(S/MARs),are critical for stabilization of transgene expression.MARs are located in close proximity to cis-acting regulatory DNA elements identified either genetically or functionally.MARs mediate structural organization of the chromatin within the nucleus.Also MARs can affect formation of eukaryotic DNA loop,maintain stability of DNA and participate in gene regulation via interaction with nuclear matrix by MAR-binding proteins.In recent years more researches have demonstrated that 5' MARs of some genes,such as chicken lysozyme gene,human β-interferon and β-globin gene,have transcriptional enhancer activity and can enhance the expression of foreign gene.5' MAR(1 600 bp) of chicken α-Globin gene is located at the boundaries of the "active" chromatin region which includes replication initiation region and nuclear matrix association region.It also contains binding sites of many transcription factors,such as SP-1,DTF-1,CTF and AP-1 et al,a long GC-rich inverse-repeated sequence GCTGCTGGCCAGCAGC and many A-boxes and T-boxes downstream of sequence.These structures are favor of the binding of nuclear matrix with MAR.GFP(green fluorescent protein) gene is a reporter gene.GFP,as expression product of GFP gene,is not only easy to observe but also easy to accurately quantify in eukaryotic expression system.So in this study GFP gene was used as a reporter gene to study the regulating effect of chicken α-globin gene 5' 1600 bp MAR on gene expression.Methods First PCR method was used to amplify 1 600 bp chicken α-Globin gene 5' MAR sequence.Then eukaryotic expression vector pGFP/2MAR was constructed by adding MAR in the upstream of CMV promoter and downstream of GFP reporter gene.Thirdly,vector pGFP/2MAR was transfected into COS7 cell line with liposome method.Finally,fluorescent microscope and flow cytometry(FCM) were used to assess GFP expression.Results From fluorescence microscope,we observed that the expression level of GFP in pGFP/2MAR transfected cells was much higher than that of pCMV/GFP transfected cells at 48 h after transfection.FCM analysis showed that the number of percentage of GFP positive cells are 17.9%(pCMV/GFP) and 33.6%(pGFP/2MAR) respectively in COS7 cells,demonstrating that MAR could significantly enhance the expression of GFP.Conclusion MAR can enhance the expression of GFP in COS7 cells, which suggests thatMAR can be further applied to improve expression efficiency of foreign gene in eukaryotic cells.But in our experiment the extent of enhancing expression of MAR is lower than those reported elsewhere.Whether this expression system has selectivity on cell line,promoter or other factors needs further study.

Key words: PCR, chicken α-Globin gene 5' MAR, pGFP/2MAR, GFP, FCM