Abstract
The recombinant baculovirus has been widely used as an efficient tool to mediate gene delivery into mammalian cells but has barely been used in fish cells. In the present study, we constructed a recombinant baculovirus containing the dual-promoter cytomegalovirus (CMV) and white spot syndrome virus (WSSV) immediate-early gene 1 (ie1) (WSSV ie1), followed by a puromycin–green fluorescent protein (Puro-GFP, pf) or puromycin–red fluorescent protein (Puro-RFP, pr) cassette, which simultaneously allowed for easy observation, rapid titer determination, drug selection, and exogenous gene expression. This recombinant baculovirus was successfully transduced into fish cells, including Mylopharyngodon piceus bladder (MPB), fin (MPF), and kidney (MPK); Oryzias latipes spermatogonia (SG3); and Danio rerio embryonic fibroblast (ZF4) cells. Stable transgenic cell lines were generated after drug selection, which was further verified by Western blot. A cell monoclonal formation assay proved the stable heredity of transgenic MPB cells. In addition, a recombinant baculovirus containing a pr cassette and four transcription factors for induced pluripotent stem cells (iPSC) was constructed and transduced into ZF4 cells, and these exogenous genes were simultaneously delivered and transcribed efficiently in drug-selected ZF4 cells, proving the practicability of this modified recombinant baculovirus system. We also proved that the WSSV ie1 promoter had robust activity in fish cells in vitro and in vivo. Taken together, this modified recombinant baculovirus can be a favorable transgenic tool to obtain transient or stable transgenic fish cells.
Highlights
The baculovirus expression system, based on the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), was mainly utilized for eukaryotic protein expression [1] or viral antigen production in host insect cells [2]
In the shuttle vectors pFastBac-CMV-ie1-pr and pFastBac-ie1-CMV-pf, the dual promoters P10 and PH of donor plasmid pFastBac-Dual were replaced by CMV and white spot syndrome virus (WSSV) ie1 promoters to drive a gene of interest, and a puromycin–red fluorescent protein (Puro-RFP, pr) or puromycin–green fluorescent protein (Puro-GFP, pf ) cassette was inserted after the WSSV ie1 or CMV promoter to act as a fluorescence reporter and drug resistance gene
A rapid way to generate stably integrated fish cell lines with large DNA capacity is required for fundamental research
Summary
The baculovirus expression system, based on the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), was mainly utilized for eukaryotic protein expression [1] or viral antigen production in host insect cells [2]. The recombinant baculovirus was used to deliver exogenous reporter genes into mammalian hepatocytes, which expanded its application in a large number of animal cells [3,4]. The application of baculovirus as a shuttle vector or gene delivery vector has mainly focused on mammalian and avian cells. Baculovirus efficiently mediates gene delivery into medaka (Oryzias latipes) embryonic stem cells [5]. It delivers the gene of interest to a particular location by injecting viruses into specific tissues directly in zebrafish (Danio rerio) [6]. The delivery of large DNA fragments to fish cells and the establishment of stably integrated cell lines via baculovirus have not yet been reported
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