title: [Construction and function identification of luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR].
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.
authors.forename: Shuo
authors.initials: S
authors.lastname: Yang
authors.forename: Jia-li
authors.initials: JL
authors.lastname: Li
authors.forename: Hui-chang
authors.initials: HC
authors.lastname: Bi
authors.forename: Shou-ning
authors.initials: SN
authors.lastname: Zhou
authors.forename: Xiao-man
authors.initials: XM
authors.lastname: Liu
authors.forename: Hang
authors.initials: H
authors.lastname: Zeng
authors.forename: Bing-fang
authors.initials: BF
authors.lastname: Hu
authors.forename: Min
authors.initials: M
authors.lastname: Huang
mesh_terms: D020413:3' Untranslated Regions; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics*; D008156:Luciferases; D000072000:NF-KappaB Inhibitor alpha; D010957:Plasmids; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha; D008156:Luciferases