Transfecting Insect Cells, Pt. 3

Howdy again, Dr. Bees here with the third part of our insect transfection series. Previously, we covered the flashBAC™ Baculovirus Expression System, and now we are covering the complementary baculovirus transfer plasmids, pOET. Insects, specifically Lepidopterans (remember in our previous post that they are the most common host of baculoviruses), have been the subject of many poets throughout history. Tennyson, Dickenson, Shelly, Wordsworth and Thoreau, to name a few. Like its sister technology, flashBAC™, pOET plasmid DNA is supplied through Mirus’s partnership with Oxford Expression Technologies.

Mirus offers three types of pOET plasmids. Each is designed to plug and play with the flashBAC™ expression system and is compatible with any baculovirus expression system relying on recombination in insect cells at the polyhedrin locus. The difference in the vectors is in the promoter regions and restriction sites, where you will cut and paste your gene of interest. Let’s check them out:

First up is pOET1, containing the AcMNPV polyhedrin promoter. Note the broad selection of restriction sites available.

Up next is pOET1C_6xHis. Nearly identical to pOET1, pOET1C_6xHis adds a C-terminal 6x His-tag fusion sequence. This is designed for purification with Ni-NTA agarose columns.

Third, the pOET6 BacMam plasmid diverges from the previous vectors by being designed for mammalian expression. This is achieved by the use of the CMV promoter. Another feature to note is the SV40 polyA signal sequence, a common terminator in mammalian plasmids.

Now that we have an idea of the tools (cell lines, expression systems, vectors), what are the applications? Baculovirus expression of mammalian proteins and viruses is unique as the producer cells are not mammalian. This has been explored in the production of vaccines and therapeutic proteins.¹ Baculovirus is also capable of infecting, but not replicating in, mammalian cells giving a potential option for cell and gene therapy.² In contrast to adeno-associated viruses (AAV), a popular virus for cell and gene therapy, the baculovirus capsid is not limited on the size of genetic payload (AAVs can only hold about 5 kb worth of nucleic acid)³ giving theoretically unlimited potential for the indications that can be targeted. In our final installment of our Transfecting Insect Cells series, we will get into some of these processes and the additional reagents Mirus offers.