title: Enhanced and prolonged baculovirus-mediated expression by incorporating recombinase system and in cis elements: a comparative study.
abstract: Baculovirus (BV) is a promising gene vector but mediates transient expression. To prolong the expression, we developed a binary system whereby the transgene in the substrate BV was excised by the recombinase (ΦC31o, Cre or FLPo) expressed by a second BV and recombined into smaller minicircle. The recombination efficiency was lower by ΦC31o (≈40-75%), but approached ≈90-95% by Cre and FLPo in various cell lines and stem cells [e.g. human adipose-derived stem cells (hASCs)]. Compared with FLPo, Cre exerted higher expression level and lower negative effects; thus, we incorporated additional cis-acting element [oriP/Epstein-Barr virus nuclear antigen 1 (EBNA1), scaffold/matrix attached region or human origin of replication (ori)] into the Cre-based BV system. In proliferating cells, only oriP/EBNA1 prolonged the transgene expression and maintained the episomal minicircles for 30 days without inadvertent integration, whereas BV genome was degraded in 10 days. When delivering bmp2 or vegf genes, the efficient recombination/minicircle formation prolonged and enhanced the growth factor expression in hASCs. The prolonged bone morphogenetic protein 2 expression ameliorated the osteogenesis of hASCs, a stem cell with poor osteogenesis potential. Altogether, this BV vector exploiting Cre-mediated recombination and oriP/EBNA1 conferred remarkably high recombination efficiency, which prolonged and enhanced the transgene expression in dividing and non-dividing cells, thereby broadening the applications of BV.
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.
authors.forename: Li-Yu
authors.initials: LY
authors.lastname: Sung
authors.forename: Chiu-Ling
authors.initials: CL
authors.lastname: Chen
authors.forename: Shih-Yeh
authors.initials: SY
authors.lastname: Lin
authors.forename: Shiaw-Min
authors.initials: SM
authors.lastname: Hwang
authors.forename: Chia-Hsin
authors.initials: CH
authors.lastname: Lu
authors.forename: Kuei-Chang
authors.initials: KC
authors.lastname: Li
authors.forename: Alice S
authors.initials: AS
authors.lastname: Lan
authors.forename: Yu-Chen
authors.initials: YC
authors.lastname: Hu
mesh_terms: D000273:Adipose Tissue / Q000166:cytology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D006224:Cricetinae; D004270:DNA, Circular / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D045170:Matrix Attachment Regions; D010012:Osteogenesis; D011817:Rabbits; D045522:Recombinases / Q000378:metabolism*; D011995:Recombination, Genetic; D018741:Replication Origin; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D019076:Transgenes
chemical_list: D004270:DNA, Circular; D036341:Intercellular Signaling Peptides and Proteins; D045522:Recombinases