High-efficiency Transient Transduction of Human Embryonic Stem Cell–derived Neurons With Baculoviral Vectors

Abstract

Transient genetic manipulation of human neurons without chromosomal integration of the transgene would be valuable but has been challenging due to the quiescent nature of these postmitotic cells. In this study, we developed a set of baculoviral vectors for transient transduction in nondividing neurons derived from human embryonic stem cells (hESCs). Using a baculoviral vector equipped with the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), we observed a quick onset of transgene expression as early as day 1 after baculoviral transduction and a high efficiency of up to 80%. Strong transgene expression in the cultured human neurons was observed for more than 1 month and the signal was easily detectable even after 3 months. Using two baculoviral vectors carrying different transgenes, we found that co-transduction at a single neuron level was possible. After transplantation into the brain of nude mice, the baculovirus-transduced human neurons were integrated into the mouse brain and maintained transgene expression for at least 4 weeks, portending the usefulness of this technique in assisting neural transplantation. Therefore, by mediating efficient transient gene expression, baculoviral vectors can provide useful tools for both basic gene function studies in human neurons and therapeutic applications of these cells.

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

“…The use of polyspecific EcTyrRS and MbPylRS facilitates the incorporation of several useful UAAs using a single suppressor virus, including UAAs with bioorthogonal handles for chemical conjugation, photoaffinity labels, and others. Baculovirus, especially the pseudotyped version, has been shown to efficiently transduce a wide range of mammalian cells both in vivo and in vitro, all of which now should be suitable hosts for UAA mutagenesis of target proteins (9)(10)(11)(12)(13)(14)(15). The unique baculovirus vectors developed in this study should also be useful for other applications, where multiple genetic elements need to be simultaneously delivered into mammalian cells.…”

“…AcNPV is also able to infect some mammalian cells, where its genetic elements remain silent rendering it replication incompetent. Thus, it can be safely used to deliver genetic cargo to a variety of different mammalian cell types both in vitro and in vivo (9)(10)(11)(12)(13)(14)(15). Several properties of baculovirus make it attractive as a potential delivery vector for the UAA incorporation machinery, including its very large cargo capacity (>30 kb), stable double-stranded DNA genome, broad host-tropism, ease of production, long shelf-life of the purified virus, intrinsically safe nature, and minimal cytotoxicity to mammalian cells, even when high multiplicity of infection (MOI) is used (9)(10)(11)(12)(13)(14)(15).…”

“…1A) that includes a VSVG expression cassette under the polyhedrin promoter, which is highly active in insect cells during the late phase of virus production but remains silent in mammalian cells. In addition, a Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was included at the 3′-UTR of the transgene, driven by the CMV-IE promoter, as this element was shown to significantly enhance expression levels and duration in mammalian cells (15). We then incorporated wild-type eGFP into this vector to generate pAcBac1-GFPwt, and generated the corresponding recombinant virus in Sf9 cells.…”

RETRACTED ARTICLE: Targeted suicide gene therapy for glioma using human embryonic stem cell-derived neural stem cells genetically modified by baculoviral vectors

“…In our previous studies, 24,25,42 a baculoviral vector with the cytomegalovirus promoter and the Glioma therapy using HSVtk-expressing NSC1 cells The HSVtk/ganciclovir(GCV) system is commonly used in suicide gene therapy for cancer. In this approach, the HSVtk gene is delivered via either direct transduction or stem cell targeting of tumors.…”

Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters

“…On this line, many viral vectors have been developed and widely used in gene transfer (transduction) and expression. To put together, they have been adenoviral (Kozarsky and Wilson 1993;Huard et al 1995), retro and lentiviral (Naldini et al 1996;Felder and Sutton 2009;Poluri et al 2003), adeno-associated viral (Hermonat and Muzyczka 1984;Carter 2005) and baculoviral (Zeng et al 2009) vectors. Among them, the baculovirus has emerged as the most promising gene delivery system in recent years (Lo et al 2009).…”