3 Methods
3.1 Generating
Pseudotyped BV
The sequence of the desired gene of interest must be known, and
can either be synthesized de novo, or amplified by PCR. The
desired target sequence must then be integrated into an acceptor
vector. Depending on which BEVS is used, different acceptor
vectors are available. To insert the desired gene sequence into the
acceptor vector, Golden Gate cloning, Gateway cloning, or classical
restriction ligation cloning can be used.
3.2 Production
of the Baculovirus
on a Bioreactor Scale
The BV is produced in Sf-9 insect cells in a stirred tank bioreactor.
All methods, such as the bioreactor assembly, the process control,
cultivation of Sf-9 cells, the initiation of cultivation, the infection of
Sf-9 cells with baculoviruses/BV, and the quantification of the BV
are described in Chap. 8 (see Note 6).
3.3 Downstream
Processing
All experiments are carried out at room temperature (RT), unless
stated otherwise.
3.3.1 Virus Harvesting
l
Differing from the protocol described in Chap. 8, the BV is the
desired product. The time of virus harvest strongly depends on
the virus concentration and functionality. Thus, it is necessary to
perform a study on time-dependent virus concentrations (vDNA
and infectious titer). Refer to the cell viability as an indication for
the time of harvest (see Note 7). Check the viability by methods
using flow cytometry or simple counting with the trypan blue
method. For reproducible results, counting should be performed in shorter intervals toward the end of cultivation (see
Note 8).
l
When not processed immediately, store the viruses under light
exclusion at 4
C, either by covering the container in foil, or by
using opaque material. Keep in mind, that the BV tends to
aggregate quickly [30]. A rapid purification reduces losses.
3.3.2 Clarification
All buffers must be sterile and filtered at 0.2 μm.
l
For filtration, the system must be sterilized. Autoclave all bottles, tubing, and connectors, as well as the Sartopure
® PP3
cartridges. Sterilization parameters for the cartridges can be
obtained in the manual.
l
Place the SARTOFLOW
® Slice 200 Benchtop System in a sterile
clean bench. Connect Sartopure
® PP3 cartridges in series, first
3 μm retention rate, followed by 0.65 μm retention rate.
l
Flush the system in total recycling mode (TRM) (see Note 9)
with 500 mL water for 10 min to wet the filters, followed by
500 mL PBS, or other physiological saline, for 10 min for
376
Friederike Eilts et al.
3.1 Generating
Pseudotyped BV
The sequence of the desired gene of interest must be known, and
can either be synthesized de novo, or amplified by PCR. The
desired target sequence must then be integrated into an acceptor
vector. Depending on which BEVS is used, different acceptor
vectors are available. To insert the desired gene sequence into the
acceptor vector, Golden Gate cloning, Gateway cloning, or classical
restriction ligation cloning can be used.
3.2 Production
of the Baculovirus
on a Bioreactor Scale
The BV is produced in Sf-9 insect cells in a stirred tank bioreactor.
All methods, such as the bioreactor assembly, the process control,
cultivation of Sf-9 cells, the initiation of cultivation, the infection of
Sf-9 cells with baculoviruses/BV, and the quantification of the BV
are described in Chap. 8 (see Note 6).
3.3 Downstream
Processing
All experiments are carried out at room temperature (RT), unless
stated otherwise.
3.3.1 Virus Harvesting
l
Differing from the protocol described in Chap. 8, the BV is the
desired product. The time of virus harvest strongly depends on
the virus concentration and functionality. Thus, it is necessary to
perform a study on time-dependent virus concentrations (vDNA
and infectious titer). Refer to the cell viability as an indication for
the time of harvest (see Note 7). Check the viability by methods
using flow cytometry or simple counting with the trypan blue
method. For reproducible results, counting should be performed in shorter intervals toward the end of cultivation (see
Note 8).
l
When not processed immediately, store the viruses under light
exclusion at 4
C, either by covering the container in foil, or by
using opaque material. Keep in mind, that the BV tends to
aggregate quickly [30]. A rapid purification reduces losses.
3.3.2 Clarification
All buffers must be sterile and filtered at 0.2 μm.
l
For filtration, the system must be sterilized. Autoclave all bottles, tubing, and connectors, as well as the Sartopure
® PP3
cartridges. Sterilization parameters for the cartridges can be
obtained in the manual.
l
Place the SARTOFLOW
® Slice 200 Benchtop System in a sterile
clean bench. Connect Sartopure
® PP3 cartridges in series, first
3 μm retention rate, followed by 0.65 μm retention rate.
l
Flush the system in total recycling mode (TRM) (see Note 9)
with 500 mL water for 10 min to wet the filters, followed by
500 mL PBS, or other physiological saline, for 10 min for
376
Friederike Eilts et al.