This month we’re celebrating the release of our newest product to come out of the OET labs – pOET9 transfer vectors. With the application of baculoviruses in mammalian protein expression becoming increasingly popular it seemed only right to introduce a new set of transfer vectors that will facilitate the production of recombinant proteins in mammalian cell lines using Autographa californica nucleopolyhedrovirus.
The vectors will combine the baculovirus expression technology with one of four unique mammalian promoters – CCAG, CMV, EF1α and SV40. The flanking sequences to each promoter enable homologous recombination with the baculovirus genome after co-transfection of insect cells. The resulting recombinant virus can be used to transduce a range of mammalian cell types and achieve expression of the target gene (figure 1) whilst preventing further virus replication; thus making it one of the safest systems on the market.
Figure 1. Relative GFP expression in CHOK1 (left) and HEK293t (right) cells transduced with recombinant baculoviruses generated using flashBAC ULTRA and pOET9 transfer vectors containing CMV, EF1α, SV40 and CCAG promoters. Expression of cells was measured by flow cytometry after 72 hours post-transduction with 150 plaque forming units per cell.
By providing a choice of gene promoter, the new transfer plasmids will allow adaptability in the strength of gene expression as well as optimisation between different mammalian cell lines (figure 2). Like all existing pOET vectors, the pOET9 range is seamlessly compatible with our flashBAC technology and a host of other homologous recombination systems.
Figure 2. Fluorescent microscopy images of HEK293t cells transduced with recombinant baculovirus generated using flashBAC ULTRA and pOET9 transfer vectors to express GFP under the following promoters A) CMV, B) EF1α, C) CCAG. Cells were infected with 150 plaque forming units per cell and observed at 72 hours post-transduction. Image D) Null virus with a CMV promoter.
Further information on each vector including sequences and plasmid maps can be found on the pOET9 products page. In addition, the recent poster displayed at the European Congress of Virology 2019 by OET scientists Dr Leo Graves and Dr Adam Chambers explains the development and applications of pOET9 during transduction of pancreatic islets with BacMAM viruses as a therapy for type 1 diabetes.