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Introduction
The use of insect cells and lytic baculoviruses for expression of full-length
mammalian proteins has been the recent method of choice for many disciplines.
Autographa californica nuclear polyhedrosis virus (AcNPV) infects
the clonal tissue culture line, Sf9, derived from Spodoptera frugiperda
insect cells. Expression of the highly abundant polyhedrin gene is non-essential
in tissue culture and its strong promoter can be used for the synthesis
of foreign gene products. The polyhedrin promoter is maximally expressed
very late in infection when the lytic virus is already killing the host
cells, giving a reasonable chance for high levels of expression even for
certain toxic proteins. Many post-translational modifications are made
correctly in insect cells and proteins unable to be expressed in E.
coli have been successfully expressed in the insect cell system.
The BD BaculoGold™ Linearized Baculovirus DNA from BD Biosciences
provides a tool for recombination efficiencies close to 100%. The principle
of this technique lies in the construction of a modified type of baculovirus
DNA, which contains a lethal deletion. This DNA does not code for viable
virus. Only co-transfection of insect cells with the viral DNA and a complementing
transfer vector construct reconstitutes viable virus. Essentially, 99%
of all virus plaques are derived from plasmid-rescued viruses which contain
and express the foreign gene from the plasmid. Since the BD BaculoGold
Linearized Baculovirus DNA contains a lacZ gene that is replaced by recombination
with the plasmid containing the foreign gene, all recombinants will produce
colorless plaques on X-gal plates. The small portion of non-recombinant
virus plaques (usually less than 1%) will stain blue on X-gal plates.
If preferred, the virus may be amplified from a single plaque from a plaque
assay.
Selection of a Transfer Vector
All polyhedrin gene locus-based baculovirus transfer vectors can be
used to rescue the lethality of the BD BaculoGold DNA. Transfer vectors
from BD Biosciences Pharmingen include pVL1392, pVL1393 (provided with
the BD BaculoGold Transfection Kit), pAcSG2, pAcGP67, pAcUW21, pAcUW51,
pAcMP2 and pAcMP3, and pAcGHLT (GST-fusion vectors) and pAcHLT (6xHis-fusion
vectors). For a complete listing and specific information about these
vectors, please visit our website at www.bdbiosciences.com. Other vectors
may also be used, but have not been tested for compatibility with the
BD BaculoGold Linearized DNA.
Recommended Controls
When performing a co-transfection to produce recombinant virus, it is
important to include both positive and negative controls. These controls
are used in all applications for producing and maintaining recombinant
baculovirus. When using the BD BaculoGold™ Linearized Baculovirus
DNA, recombinant baculovirus expressing the XylE protein can be used as
a positive control and are generated by performing a co-transfection with
the pVL1329-XylE Baculovirus Control Plasmid, Cat. No. 554807. Cells infected
with this recombinant virus express recombinant XylE and turn yellow in
the presence of the substrate catechol. Alternatively, recombinant virus
can be generated using the BD BaculoGold Bright Linearized Baculovirus
DNA which contains the GFP gene, expressed constitutively. The GFP protein
is fluorescent and cells generating virus expressing this protein will
glow green and can be assayed or sorted using flow cytometry or visualized
by fluorescence microscopy. Recombinant virus can be titered or purified
using these applications. Uninfected Sf9 cells are used as the negative
control. Uninfected cells continue to proliferate; therefore, relative
cell number is a criterium for degree of infection. The use of both positive
and negative controls is particularly valuable for the end-point dilution
and plaque assays.
Material Required
MATERIALS AND REAGENTS
Transfection Reagents:
BD BaculoGold Transfection Kit, Cat. No. 560129, contains:
Linearized Baculovirus DNA 2.5 µg
pVL1392 Baculovirus Transfer Vector 5 µg
pVL1393 Baculovirus Transfer Vector 5 µg
pVL1392-XylE Baculovirus Control Vector* 5 µg
Transfection Buffer A 5 ml
Transfection Buffer B 5 ml
or
BD BaculoGold Linearized Baculovirus DNA, Cat. No. 554739
Transfection Buffer A&B Set, Cat. No. 554806
Transfer Vector of choice
TNM-FH Insect Cell Medium, Cat. No. 554760
Sf9 cells in TNM-FH Medium, Cat. No. 554763 (grow to log-phase, at least
98% viable)
1 ml, 5 ml, 10 ml, 25 ml and/or 50 ml sterile pipets
1.5 ml sterile microcentrifuge tube (or comparable)
20 µl and 1000 µl sterile pipet tips
50 ml sterile conical tube, BD Falcon™ Cat. No. 352098
15 ml sterile conical tube, BD Falcon™ Cat. No. 352097
Tissue Culture Dish, 60 x 15 mm, BD Falcon™ Cat. No. 353802
Tissue Culture Dish, 150 x 25 mm, BD Falcon™ Cat. No. 352097
*Catechol, Sigma, Cat. No. C-9510 (When using pVL1392-XylE Control Vector)
EQUIPMENT
Sterile laminar flow hood
Electric pipettor
L 2, L 20 and L 1000 Pipettors
Mini centrifuge
27°C Incubator
Light microscope
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