Construction of luciferase reporter plasmids of rat S100A8 promoter and initial identification of SOX7 binding element
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    Abstract:

    Objective:This study aims to construct luciferase reporter plasmids of rat S100 calcium binding protein A8(S100A8) gene promoter and detect their activity in HEK293T cells in response to SRY-box transcription factor 7(SOX7)overexpression,meantime screen the possible binding elements for SOX7. Methods:Rat S100A8 gene full length promoter was amplified by PCR and cloned into the luciferase reporter plasmid(pGL3basic),and named pGL3S100A8FL. The plasmid of pGL3S100A8FL and previously constructed plasmid of pIRES2-SOX7 were co-transfected into HEK293T cells,and then the luciferase activity was detected. Meanwhile,the potential SOX7-binding elements within S100A8 promoter were predicted by JASPAR. Based on the predicted results,four plasmids of truncated S100A8 gene promoter(pGL3S100A8 1~4)were co nstructed. The plasmids of pGL3S100A8FL or pGL3-S100A8 1~4 and pIRES2SOX7 were cotransfected into HEK293T cells respectively. Then,the luciferase activity was detected. Next,S100A8 gene promoter of SOX7-binding element(-86~-57 nt)mutated plasmid was constructed(pGL3-S100A8-M). The HEK-293T cells were transfected with pGL3-S100A8-M and pIRES2-SOX7 plasmid,and the luciferase activity was detected. Results:The plasmids of pGL3S100A8FL and pIRES2SOX7 were co transfected into HEK293T cells,found that the luciferase activity of S100A8 gene promoter was markedly increased in response to SOX7 overexpression. The plasmids of pGL3S100A8FL or pGL3S100A8 1~4 and pIRES2SOX7 were co transfected into HEK293T cells,and the result displayed that the activity of pGL3S100A8-4 was much lower than that in pGL3S100A8-FL and pGL3-S100A8-1~3,indicating that the region of rat S100A8 promoter(-200~+51 nt)might contain a SOX7-binding element(-86~-57 nt). Then the -86~-57 nt mutated plasmid(pGL3S100A8M) or pGL3S100A8FL and pIRES2SOX7 were co transfected into HEK293T cells,and the result revealed that the activity of pGL3S100A8-M was much lower than that of pGL3S100A8FL,indicating that the SOX7 may bind to the element of rat S100A8 gene promoter -86~-57 nt. Conclusion:The rat full length and truncated rat S100A8 promoter luciferase reporter plasmids were constructed successfully,and the possible SOX7 binding element was preliminary determinated.

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彭明玉,何庆玲,王文博,赵 聃,张 婧,王迎伟,邱 文.大鼠S100A8基因启动子质粒的构建及其SOX7结合元件的初步鉴定[J].南京医科大学学报(自然科学版英文版),2021,(5):637-642.

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  • Received:October 17,2020
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  • Online: June 02,2021
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