title: Construction of a lentiviral T/A vector for direct analysis of PCR-amplified promoters.
abstract: The promoter plays an important role in the regulation of gene expression. To analyze a promoter's activity, we developed a novel lentiviral T/A vector that contains two reporter genes, a luciferase (Luc2) gene and a green fluorescent protein (Venus) gene, that are linked via an internal ribosome entry site (IRES2). To test the performance of this vector, phosphoglycerate kinase-1 (PGK) and elongation factor-1α (EF1α) promoters were amplified by PCR and inserted into this lentiviral T/A vector using T4 DNA ligase, yielding two promoter-reporter vectors: pLent-T-PGK and pLent-T-EF1α. When these vectors were transfected into 293T cells, we observed a higher level of Venus expression under a fluorescence microscopy in the case of pLent-T-EF1α as compared to pLent-T-PGK. The results of the luciferase reporter assay showed that the ratio of the promoter activities of EF1α and PGK was approximately 9:1. The two promoter-reporter vectors were also packaged as lentiviral particles to conduct promoter activity assay in cultured cells. The ratio of the promoter activities of EF1α and PGK was 4.23:1 when they were infected into 293T cells at a multiplicity of infection of 1. This value is comparable to that of a parallel experiment using the commercial luciferase reporter vector pGL4.10 with an activity ratio of 5.99:1 for EF1α and PGK. These results indicate that lentiviral T/A vector will be a useful tool for analysis of promoter activity and specificity.
authors.affiliation: Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agriculture Science, Hangzhou, China.
authors.forename: Fu-xian
authors.initials: FX
authors.lastname: Yu
authors.forename: Zhi-wei
authors.initials: ZW
authors.lastname: Zhu
authors.forename: Xiao-yu
authors.initials: XY
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Huang
authors.forename: Tuan-yuan
authors.initials: TY
authors.lastname: Shi
authors.forename: Jun-xing
authors.initials: JX
authors.lastname: Li
authors.forename: Jian-zhi
authors.initials: JZ
authors.lastname: Pan
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D008156:Luciferases / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics*; D008856:Microscopy, Fluorescence; D020648:Peptide Elongation Factor 1; D010735:Phosphoglycerate Kinase / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014162:Transfection / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; D020648:Peptide Elongation Factor 1; C054562:yellow fluorescent protein, Bacteria; D008156:Luciferases; C577282:PGK1 protein, human; D010735:Phosphoglycerate Kinase
references.citation: Biochem Biophys Res Commun. 2006 Mar 31;342(1):273-9
references.pmid: 16480950
references.citation: Science. 2006 May 26;312(5777):1215-7
references.pmid: 16728641
references.citation: Genes Dev. 2010 Sep 1;24(17):1882-92
references.pmid: 20810647
references.citation: Oncogene. 2010 Nov 25;29(47):6245-56
references.pmid: 20802531
references.citation: Science. 1996 Apr 12;272(5259):263-7
references.pmid: 8602510
references.citation: Am J Physiol Endocrinol Metab. 2001 Dec;281(6):E1128-36
references.pmid: 11701425
references.citation: Mol Biol (Mosk). 2007 Jul-Aug;41(4):719-24
references.pmid: 17936994
references.citation: Mol Biol Rep. 2010 Jul;37(6):2757-65
references.pmid: 19757163
references.citation: Development. 1996 Aug;122(8):2415-25
references.pmid: 8756287
references.citation: Plasmid. 2002 Sep;48(2):160-3
references.pmid: 12383733
references.citation: Nucleic Acids Res. 1991 Mar 11;19(5):1156
references.pmid: 2020554
references.citation: Theriogenology. 2007 Jan 1;67(1):127-33
references.pmid: 17070901
references.citation: Curr Issues Mol Biol. 2000 Jan;2(1):1-7
references.pmid: 11464915
references.citation: Anal Biochem. 1994 Jan;216(1):235-6
references.pmid: 8135360
references.citation: J Biol Chem. 2004 Feb 20;279(8):7275-86
references.pmid: 14625278
references.citation: Nucleic Acids Res. 1988 Oct 25;16(20):9677-86
references.pmid: 2460825
references.citation: Biochem Biophys Res Commun. 2008 Jul 4;371(3):546-50
references.pmid: 18448070
references.citation: Mol Vis. 2007 May 17;13:730-9
references.pmid: 17563724
references.citation: J Virol. 1998 Oct;72(10):8150-7
references.pmid: 9733856
references.citation: PLoS One. 2010 May 12;5(5):e10611
references.pmid: 20485554
references.citation: Mol Biotechnol. 1999 Oct;12(3):281-3
references.pmid: 10631685
references.citation: J Clin Invest. 2010 Jan;120(1):379-89
references.pmid: 20038801
references.citation: DNA Cell Biol. 1993 Oct;12(8):763-70
references.pmid: 8397833
references.citation: Biochem Biophys Res Commun. 2012 Jun 22;423(1):128-33
references.pmid: 22634312