首页|Synthetic biology approach to developing all-in-one baculovirus vector using mammalian introns and miRNA binding sites
Synthetic biology approach to developing all-in-one baculovirus vector using mammalian introns and miRNA binding sites
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NSTL
Elsevier
Background: Insect baculovirus is a promising vector for gene delivery into mammalian cells. We developed a Cre/loxP-based hybrid baculovirus comprising two viruses: one expressing Cre recombinase and the other harboring the transgene flanked by two loxP sites. Co-transduction of mammalian cells with two viruses confers Cre expression, which excises the loxP-flanking cassette off baculovirus genome and catalyzes DNA minicircle formation, thereby prolonging transgene expression. Two separate baculoviruses avoid undesirable recombination and loss of transgene during virus production process, but reduces gene delivery efficiency and complicates applications. Methods: To tackle this problem, we exploited synthetic biology strategy to control Cre expression and develop an all-in-one baculovirus harboring both cre and loxP-flanking transgene cassette. We controlled cre transcription with a mammalian EF-1 alpha promoter, and regulated cre translation with an intron inserted within the coding region and bantam miRNA binding site at the 3'UTR. Significant findings: The all-in-one baculovirus vector selectively conferred Cre/loxP-mediated recombination in mammalian cells, but not in E. coli and Sf-9 cells, hence circumventing transgene excision from the virus genome during gene cloning in E. coli and virus amplification in insect cells. The all-in-on vector enabled formation of DNA minicircle in mammalian cells at efficiencies exceeding 80% and implicated its potential for gene delivery. Our design may be expanded to other gene delivery systems that require two separate vectors and is beneficial to gene therapy applications. (C) 2021 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
NSTL
Elsevier
