HR system is an easy, fast and safe system to generate recombinant baculovirus
In order to evaluate the HR system for the generation of recombinant baculoviruses intended for rAAV vector production, we first transfected Sf9 insect cells with the donor plasmid pBac. The plasmid pBac-rep/cap carries the AAV2 rep and AAV8 cap ORFs that were optimized for the expression in Sf9 cells. This plasmid was digested (linear) or not (circular) with restriction enzymes on both sides of the GOI. Indeed, we hypothesized that a linearized donor plasmid would be more prone to provide efficient homologous recombination as suggested in previous genome editing report (Song and Stieger, 2017). We collected and amplified five individual clones per condition to be further characterized by assessing the number of infectious particles. The circular pBac leads to heterogenous clones with low infectivity (<5.0 x 108 IU/ml) (Fig. 2a ). Inversely the linear donor plasmid produced clones with high infectivity with a mean of 9.5 ± 1.1 x 108 infectious unit (IU)/ml. Interestingly, chloramphenicol coding sequence was still detectable by PCR amplification in the cleared supernatant for the circular plasmid meaning that the parental bacmid contaminates the P1 baculovirus stock, contrary to the linear plasmid condition (data not shown). Next, the genetic stability of the recombinant baculovirus was analyzed for each clone by two qPCR assays targeting the AAV Rep sequence (insert) and the baculovirus DNA polymerase gene (baculovirus backbone). The results are represented as a ratio “bac/rep” (Fig. 2b ). The ratio insert/backbone was close to 1 in all clones produced with the linear plasmid, confirming that all five baculoviral clones carried the exogenous cassette “rep2/cap8”. We also evaluated the functionality of the cassette “rep2/cap8” to express AAV proteins in Sf9 cells by Western blot analysis for each P1 clone. The expression of Rep78, Rep52, and VP1, VP2, VP3 proteins was detected only for baculoviruses derived from the linear pBac plasmid (Fig. 2c ) and was stable over 10 serial passages. Indeed, no signal was detected for clones derived from circular pBac plasmid confirming the absence of the expression casette in the baculovirus population. Altogether, these observations suggested that is preferable to use linear donor template to favor HR efficiency, and drastically reduce the proportion of DI baculoviruses.