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.