Transfecting Insect Cells, Pt. 2

From Webster’s dictionary (emphasis mine):

flash•back
/ˈflaSHˌbak/
noun
1. interruption of chronological sequence (as in a film or literary work) by interjection of events of earlier occurrence
2. a past incident recurring vividly in the mind

The flashBAC™ Baculovirus Expression System is an expression vector designed from the genome of Autographa californica multiple nucleopolyhedrovirus  (AcMNPV). A. californica, the alfalfa looper, is a Noctuidae moth related to our friends S. frugiperda and T. ni from “Transfecting Insects, Pt. 1.” AcMNPV is a member of the Baculoviridae family of viruses which have broad host infectivity, but a full lifecycle only in insect cells.¹

Let’s look at our flashBAC™ vector in detail–the flashBAC™ vector, like most baculovirus expression vectors, is based off the AcMNPV genome and contains several deletions to allow for and to improve recombinant gene expression.² The polyhedrin gene is one such deletion. Though the polyhedrin coding sequence is interrupted, the polyhedrin gene promoter is retained to drive strong gene expression of foreign genes inserted downstream. The original flashBAC™ DNA also contains deletion of the chitinase gene, which is thought to improve recombinant protein stability by modulating the secretory pathway of insect cells.³ The original flashBAC™ vector is suitable for expression of most nuclear or cytoplasmic proteins. However, if you’re looking to express more “difficult” constructs, like membrane proteins or secreted proteins, using the flashBAC™ ULTRA vector may be even better. flashBAC™ ULTRA DNA has additional deletions of the cathepsin, p10, p26 and p74 genes, which were found to further enhance recombinant protein production in some cases.⁴

How do you use flashBAC™ to express your gene of interest (GOI)? flashBAC™ DNA contains a bacterial artificial chromosome (BAC) at the polyhedrin gene locus that is flanked by lef2 and a partial copy of orf1629. See the Figure below.

The BAC sequence enables propagation of flashBAC™ DNA in bacteria, while the lef2 and orf1629 sequences are used for homologous recombination with a plasmid containing your GOI, which is also flanked by lef2 and orf1629 sequences. The plasmid containing your GOI is sometimes called a ‘transfer vector.’ Importantly, homologous recombination occurs in insect cells that are co-transfected with the flashBAC™ DNA and the transfer vector containing your GOI.

The recombination event serves two purposes: 1) it results in the replacement of the BAC with your GOI, and 2) it prevents “parental” baculovirus, i.e. baculovirus not carrying your GOI, from being produced. An intact orf1629 gene is required for baculovirus production, and orf1629 is only made complete if homologous recombination between flashBAC™ DNA and the transfer vector is successful during co-transfection in insect cells. Co-transfection is easy. After co-transfection, harvest your baculovirus carrying your GOI a couple of days later. No bacterial transformations or plaque purification required!

The lef2 and orf1629 sequences flank the BAC and GOI in their respective vector DNA (DNA being the literary material of life). The parental virus is replication deficient via interjection of a deletion in orf1629. 

Did you have a flashBAC™? Contact Mirus Bio Technical Support (techsupport@mirusbio.com) for more information about the flashBAC™ Baculovirus Expression System.