Figure 1: Gene transfer systems to generate recombinant
baculoviruses.
a) Tn7 (Bac-to-Bac®) and HR (BacMagic-2 or
flashBAC GOLD) bacmid systems are derived from wild-type circular
baculoviral genome by deletion of structural polyhedrin gene
(Ac8) and insertion of bacterial artificial chromosome (BAC) as
described by Luckow et al., 1993 and Possee et al., 2008. Commercial HR
systems are optionally deleted for accessory genes, i.e.chitinase (Ac126) and/or v-cathepsin (Ac127) which are of
interest for our study. b) Tn7 system is handled in bacteria
where both “pFastBac” donor plasmid and Tn7-bacmid DNA are maintained
and amplified thanks to the selective genes coding for ampicillin (AmpR)
and gentamycin (GmR). Tn7 transposition is induced with the help of
pMON7124 helper plasmid that expresses transposition proteins
(TnsABCD ) in trans leading to gene of interest (GOI)
transfer from the donor plasmid pFastBac to the Tn7-bacmid DNA by attTn7
site recognition, including the selective gene coding for GmR. The
presence of the expression cassette into the Tn7-bacmid DNA is
characterized by PCR, before to be purified from bacteria and then
transfected into Sf9 insect cell line, where the first BEV progeny is
generated (P0). Viral clones are isolated by plaque assay (P1) and
characterized (i.e. titer, sequence identity and genetic stability). One
stable clone meeting specification is then amplified to generate a
master viral seed (P2 or P3). HR system, “pBac” donor plasmid and the
HR-bacmid DNA are directly co-transfected in Sf9 insect cell line,
without the need of a bacterial step. Homologous recombination can occur
by homology arms recognition of lef2 and non-deletedorf1629 sequences from the donor plasmid up-to the HR-bacmid DNA.
The cassette is transferred into the bacmid in place of the existing BAC
sequence carrying the Mini-F, and the selective gene
chloramphenicol (CmR), that are finally deleted from the final
construct. Moreover, the orf1629 sequence is fully reconstituted
leading to viable BEV progeny generation (P0). Similarly, to Tn7 system,
one clone (P1) is then characterized and amplified up to MVS build-up
(P2 or P3).