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Construction and function identification of luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR / 药学学报
Acta Pharmaceutica Sinica ; (12): 80-85, 2016.
Article in Chinese | WPRIM | ID: wpr-320013
ABSTRACT
This study aims to investigate the function of two SNPs (rs8904C > T and rs696G >A) in 3' untranslated region (3'UTR) of NFKBIA gene by constructing luciferase reporter gene. A patient's genomic DNA with rs8904 CC and rs696 GA genotype was used as the PCR template. Full-length 3'UTR of NFKBIA gene was amplified by different primers. After sequencing validation, these fragments were inserted to the luciferase reporter vector, pGL3-promoter to construct recombinant plasmids containing four kinds of haplotypes, pGL3-rs8904C/rs696G, pGL3-rs8904C/rs696A, pGL3-rs8904T/rs696G and pGL3-rs8904T/rs696A. Then these plasmids were transfected into LS174T cells and the luciferase activity was detected. Compared with pGL3-vector transfected cells (negative control), the luciferase activity of the four kinds of recombinant plasmids was significantly decreased (P < 0.001). For rs696G > A, the luciferase activity of the recombinant plasmids containing A allele (pGL3-rs8904C/rs696A and pGL3-rs8904T/rs696A) was about 45.1% (P < 0.05) and 56.1% (P < 0.001) lower than those containing G allele (pGL3-rs8904C/rs696G and pGL3-rs8904T/rs696G), respectively. For rs8904C > T, there were no significant differences in the luciferase activity between the recombinant plasmids containing T allele and those with C allele. Together, the luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR were constructed successfully and rs696G > A could decrease the luciferase activity while rs8904C >T didn't have much effect on the luciferase activity.
Subject(s)
Full text: Available Index: WPRIM (Western Pacific) Main subject: Plasmids / Transfection / Promoter Regions, Genetic / Genes, Reporter / 3&apos; Untranslated Regions / I-kappa B Proteins / Polymorphism, Single Nucleotide / NF-KappaB Inhibitor alpha / Genetic Vectors / Genetics Type of study: Diagnostic study Limits: Humans Language: Chinese Journal: Acta Pharmaceutica Sinica Year: 2016 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Plasmids / Transfection / Promoter Regions, Genetic / Genes, Reporter / 3&apos; Untranslated Regions / I-kappa B Proteins / Polymorphism, Single Nucleotide / NF-KappaB Inhibitor alpha / Genetic Vectors / Genetics Type of study: Diagnostic study Limits: Humans Language: Chinese Journal: Acta Pharmaceutica Sinica Year: 2016 Type: Article