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. 2020 Apr 17;11(4):439. doi: 10.3390/genes11040439

Figure 1.

Figure 1

Construction of baculoviral transfer plasmids for vector production in Sf9 cells and the plasmids for rAAV2/HBoV1 vector production in HEK293 cells. (A) BEVs for rAAV2/HBoV1 production. Schematically diagrammed are structures inside the BEVs that were involved in rAAV2/HBoV1 production. Bac-AAV2ITR-GFP-Luc carries an rAAV2 genome of 5.4-kb; Bac-AAV2Rep-HBoV1Cap expresses AAV2 Rep proteins and HBoV1 capsid proteins as shown; and Bac-HBoV1NP1 expresses HBoV1 NP1. P10 and Ph are baculoviral promoters, and CMV and SV40 are cytomegaloviral immediate early and SV40 virus early promoters, respectively. PolyA: polyadenylation signal; Luc: firefly luciferase. (B) Plasmids used for vector production in HEK293 cells. pAAV2ITR-GFP-Luc carries the same rAAV2 genome as shown in panel A. pCMVNS*Cap-P5Cap is a two-in-one plasmid. It was derived from the plasmid pHBoV1CMVNS*Cap [28], in which the NS1/2 ORF was early terminated. An AAV2 P5 and P19 driven rep gene was cloned after the CMV promoter-driven HBoV1 cap gene expression cassette. (C,D) Codon optimization. Both wild type (wt) and optimized (opt.) sequences between ATGs of the AAV2 Rep78 and Rep52 ORFs (C) and of the HBoV1 VP1 and VP3 ORFs (D) are diagrammed. Nucleotides in red indicate mutations. (E) BEVs for rAAV2 production in Sf9 cells. Bac-AAV2ITR-GFP carries a GFP expression cassette under both the CMV and P10 promoters. Bac-AAV2Rep-Cap carries expression cassettes of AAV2 cap and AAV2 rep under the P10 and Ph promoters, respectively.