Efficient and Stable Delivery of Multiple Genes to Fish Cells by a Modified Recombinant Baculovirus System

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.

Virus susceptibility of a new cell line derived from the fin of black carp Mylopharyngodon piceus

“…Third, MPF is an ideal tool used for the expression of exogenous DNA. Generally, the transfection efficiency of exogenous DNA in fish cells is much lower when compared with mammalian cells (Falco et al ., ; Ruiz et al ., ; Wang et al ., ). In this study, the transfection efficiency of MPF reached 23% using a CUY21EDIT II electroporator.…”

“…CUY21EDIT II generates two pulses, poration pulses which make small holes in the surface of the cell and driving pulses which move DNA into the inside of the cell. The direct electroporation of DNA into MPF cells produced both good cell viability and consistently high transfection rates, which was higher than that in Megalobrama amblycephala fin (MAF; Zhu et al ., ), Sahul Indian grouper eye (SIGE; Parameswaran et al ., ), black carp kidney (MPK; Xue et al ., ) and zebrafish embryo fibroblast (ZF4; Wang et al ., ). Moreover, stable transgenic MPF cells were also established.…”

“…The proportion of fluorescence positive cells was counted with a haemocytometer. To establish stable transgenic cells, the baculovirus system expressing red fluorescent protein was applied and transgenic cells were selected by 1 μg ml −1 of puromycin for 2 weeks (Wang et al ., ).…”

“…Cytopathic effects (CPE) were observed under an optical microscope. The 50% tissue culture infectious dose (TCID50) was calculated accordingly (Reed & Muench, ; Wang et al ., ). Moreover, different viral titres of SVCV were added to MPF (10 6 TCID50) or FHM (10 5 TCID50) in 12‐well plates to examine the CPEs and transcripts of a viral gene.…”

“…The fathead minnow cell line (FHM) and SVCV were maintained in the lab. Viral titre and susceptibility of MPF to SVCV were tested as previously described (Shao et al ., ; Wang et al ., ; Xue et al ., ). The virus was serially diluted by from 10 −1 to 10 −13 in basic medium.…”