HR- and Tn7-mediated systems operate with an artificial bacterial chromosome (BAC) integrated in baculoviral DNA
The generation of BEVs by Tn7 transposition in bacteria is a well described method in the literature and is commercialized as Bac-to-Bac system (Bac-to-Bac, 2015, ThermoFisher Scientific). The circular baculoviral DNA derived from theAutographa californica nucleopolyhedrovirus (AcMNPV) has been engineered to insert a bacterial artificial chromosome (BAC) into thepolyhedrin locus (Smith et al., 1983) that contains the kanamycin selective gene (KnR), the mini-F bacterial replicon and a mini-attTn7 site inserted into the LacZα region (Luckow et al., 1993) (Fig. 1a ). This shuttle vector named “bacmid” can easily be modified and amplified using conventional E. coli bacterial transformation in which a helper plasmid (pMON7124) provides the functions requiredin trans for Tn7 transposition. The bacmid with DNA insert is selected under gentamycin (GmR) antibiotic pressure (Fig. 1b ). More recently, an alternative system to Tn7 transposition has emerged (Possee et al., 2008; Zhao, 2003) based on homologous recombination (HR) mechanism and inspired from a previous study (Kitts and Possee, 1993). The bacmid used for the generation of BEV by HR has been commercialized as BacMagic (Merck Millipore, Novagen) or flashBAC systems (Oxford Expression Technologies). In these systems, HR system does not require the use of bacterial step to perform gene cloning into baculovirus backbone, which is laborious and time-consuming. Nonetheless, the parental bacmid contains a chloramphenicol resistance (CmR) gene to allow its amplification and selection in bacteria. By directly co-transfecting insect cell line with the defective parental bacmid DNA (orf1629- ) and a donor plasmid containing the gene of interest (GOI) flanked by homologous sequences lef2 and non-deleted orf1629 , HR events induce bacmid knock-out (KO) of the bacterial replicon from the targeted polyhedrin locus and knock-in (KI) of the GOI that simultaneously restores the orf1629 entire sequence allowing BEV genome replication and the generation of viable recombinant baculoviruses progeny named P0 seed stock (Fig. 1b ).
Whatever the system used to generate the P0, Tn7 or HR, one round of viral plaque isolation is required to eliminate DIs, then to amplify and characterize few P1 clones. In this work, five P1 clones have been selected per construct. Finally, one clone that meets the required specifications, i.e. sequence identity and genetic stability, will be selected and used to generate the master viral seed P2 and P3 (Fig. 1b ). Importantly, commercial sources for the HR system also offer bacmid versions that are deleted in some “accessory” genes. In this study, HR-derived BEV devoid of chitinase (AcORF-126,chiA ) and cathepsin (AcORF-127, v-cath ) genes were used. The two encoded proteases favor postmortem liquefaction of the larvae and release of occlusion derived viruses in the environment (Hawtin et al., 1997) but are non-essential for baculovirus replication in vitro (Fig. 1a ). Several AAV capsids, including 1, 3, 6, 7, 8 and rh10, have been shown to be susceptible to the baculovirus cathepsin (Galibert et al., 2018). In particular, the subsequent VP1/VP2 cleavage of serotype 8 capsid has been documented to impair in-vivoinfectivity. The sequences of both capsid variants, AAV8 and AAVanc80, used in this study contain the major predicted cathepsin cleavage site.