title: Construction and gene expression analysis of a single-stranded DNA minivector based on an inverted terminal repeat of adeno-associated virus.
abstract: The plasmid vectors currently used for nonviral gene transfer have the disadvantage of carrying a bacterial backbone and an antibiotic resistance gene, which may cause side effects. The adeno-associated virus (AAV) genome is a linear single-stranded DNA (ssDNA) molecule with palindromic inverted terminal repeat (ITR) sequences forming double-stranded DNA (dsDNA) hairpin (HP) structures at each end. Based on the AAV genome, we constructed an AAV-ITR ssDNA minivector that consists of a GFP expression cassette flanked by both ITR sequences of 125 nucleotides. The minivectors were produced by digestion of the parental plasmids followed by denaturation. The self-complementary inverted T-shaped HP structure of the minivector was automatically formed. The HEK 293T cells were transfected with the AAV-ITR ssDNA minivector, plasmid, and dsDNA expression cassette. The results showed that AAV-ITR ssDNA minivector had relatively low gene expression efficiency in vitro. However, we found that the GFP expression efficiency of the D sequence-deleted AAV-ITR ssDNA minivector was significantly increased and was similar to those obtained with the plasmid and dsDNA expression cassette. Our data suggest that the AAV-ITR ssDNA minivector may be a new type of gene expression vector for gene therapy besides the virus and plasmid.
authors.affiliation: Key Lab of Bio-Medical Diagnostics, CAS, Institute of Biomedical Engineering and Technology, CAS, No. 88 Keling Road, Suzhou New District, Suzhou, 215163, People's Republic of China.
authors.forename: Han
authors.initials: H
authors.lastname: Ping
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Liu
authors.forename: Dongqin
authors.initials: D
authors.lastname: Zhu
authors.forename: Taiming
authors.initials: T
authors.lastname: Li
authors.forename: Chun
authors.initials: C
authors.lastname: Zhang
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004277:DNA, Single-Stranded / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D020079:Terminal Repeat Sequences / Q000235:genetics*; D014162:Transfection
chemical_list: D004277:DNA, Single-Stranded; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins