title: A comparative analysis of constitutive promoters located in adeno-associated viral vectors.
abstract: The properties of constitutive promoters within adeno-associated viral (AAV) vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase), were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine Nagakute, Aichi, Japan.
authors.forename: Lkhagvasuren
authors.initials: L
authors.lastname: Damdindorj
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine Nagakute, Aichi, Japan.
authors.forename: Sivasundaram
authors.initials: S
authors.lastname: Karnan
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine Nagakute, Aichi, Japan.
authors.forename: Akinobu
authors.initials: A
authors.lastname: Ota
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine Nagakute, Aichi, Japan.
authors.forename: Ekhtear
authors.initials: E
authors.lastname: Hossain
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine Nagakute, Aichi, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Konishi
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine Nagakute, Aichi, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Hosokawa
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine Nagakute, Aichi, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Konishi
mesh_terms: D045744:Cell Line, Tumor; D019941:Cyclin-Dependent Kinase Inhibitor p16; D003587:Cytomegalovirus / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D045325:HCT116 Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic
chemical_list: C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004279:DNA, Viral; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C080086:phosphatidylinositol glycan-class A protein; D049452:Green Fluorescent Proteins