Construction and function identification of luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR.
- Author:
Shuo YANG
;
Jia-li LI
;
Hui-chang BI
;
Shou-ning ZHOU
;
Xiao-man LIU
;
Hang ZENG
;
Bing-fang HU
;
Min HUANG
- Publication Type:Journal Article
- MeSH:
3' Untranslated Regions;
Genes, Reporter;
Genetic Vectors;
Humans;
I-kappa B Proteins;
genetics;
Luciferases;
NF-KappaB Inhibitor alpha;
Plasmids;
Polymorphism, Single Nucleotide;
Promoter Regions, Genetic;
Transfection
- From:
Acta Pharmaceutica Sinica
2016;51(1):80-85
- CountryChina
- Language:Chinese
-
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.
