Construction and function identification of luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR
10.16438/j.0513-4870.2015-0611
- VernacularTitle:NFKBIA 3'UTR不同基因型载体的构建及其功能差异性比较
- Author:
Shuo YANG
1
;
Jia-li LI
1
;
Hui-chang BI
1
;
Shou-ning ZHOU
1
;
Xiao-man LIU
1
;
Hang ZENG
1
;
Bing-fang HU
1
;
Min HUANG
1
Author Information
1. Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
- Publication Type:ORIGINAL ARTICLES
- Keywords:
NFKBIA;
3' untranslated region;
single-nucleotide polymorphism;
luciferase reporter gene vector;
rs696;
rs8904
- From:
Acta Pharmaceutica Sinica
2016;51(1):80-
- 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.
