From the Eye Research Institute of Canada, Toronto,
Ontario M5T 2S8 and the § Department of Medical Genetics and
Microbiology and
Departments of Ophthalmology and Laboratory
Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G
1L5, Canada
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ABSTRACT |
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Semliki Forest virus (SFV) vectors can be produced faster, and have a wider host range, than baculovirus vectors. However, the original SFV system requires in vitro manipulation of RNA. We have generated a system that is wholly DNA-based. Both the replicon vector, encoding SFV polymerase and the protein of interest, and the helper vector, encoding viral structural proteins, were modified so that expression was RNA polymerase II-dependent. Transfection of the modified replicon plasmid alone generated 20-30-fold more protein than obtained from a simple expression vector. Expression required the SFV replicase, which amplifies replicon RNA. The SFV-based vector generated 10-20-fold more protein than a plasmid based on Sindbis virus. Cotransfection of SFV replicon and helper vectors generated viral titers of around 106 infectious particles/ml. A single electroporation, plated on one 10-cm plate, generated enough virus (107 particles) to produce >500 µg of protein. Wild type, replication proficient virus was not detected in three tests utilizing almost 108 viral particles, a distinct advantage over a DNA Sindbis-based system in which over half the virus particles generated are fully infectious. The new SFV vectors significantly enhance the utility of this expression system.
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INTRODUCTION |
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A fundamental technique in both basic and applied molecular biology is the production of heterologous proteins. Bacterial systems are widely utilized, but are hampered by the inability to modify eukaryotic proteins appropriately. In addition, eukaryotic cells have evolved specific mechanisms, which are lacking in bacteria, to fold multidomain proteins (1). Bacterial hosts, therefore, are considerably less efficient at producing well folded, soluble, biochemically active protein than mammalian cells.
Gene expression in mammalian culture is impeded by poor transfection efficiency, limited host cell range, and the complexity of the expression system. Recombinant viruses are the most efficient tools for protein production in higher eukaryotic cells. Baculovirus vectors can produce large amounts of heterologous protein, but utilize insect host cells, which have been shown to glycosylate proteins differently than mammalian cells. The baculovirus and vaccinia systems require screening of viruses generated by homologous recombination (2, 3), which can be time-consuming, especially if large numbers of genetic mutants are required.
To overcome these obstacles, alphavirus vectors have emerged as useful tools in heterologous protein production. The Sindbis virus and Semliki Forest virus (SFV),1 members of the Togaviridae family, are the best studied alphaviruses (4). Both have been harnessed into gene expression systems because of their self-amplifying genomes that require only the host translational machinery to replicate (5, 6).
In its normal replication cycle, SFV infects the cell and replication
commences with translation of the 5' two-thirds of the positive-sense
genomic RNA into a polyprotein, with subsequent autoproteolytic
cleavage into four non-structural proteins (nsPs), nsP1 to nsP4,
forming an RNA replicase (Fig. 1A). This complex targets a
replication sequence at the 3' end of the RNA genome, and replicates
full-length ()-strand RNA from (+)-strand genomic RNA and vice
versa. The structural proteins (sPs) required for a mature virion
are encoded by the last one-third of the SFV genome (Fig.
1A). This region is transcribed into a sub-genomic message by the SFV replicase, and translated into a polyprotein that is autoproteolytically cleaved, producing the capsid protein and two
envelope glycoproteins (Fig. 1A). The capsid protein
recognizes a packaging signal buried in the coding region for nsP2 and,
together with the full-length RNA, forms a nucleocapsid (7-9). This
structure interacts with the envelope proteins' cytoplasmic domains
and buds from the plasma membrane to form a mature, infectious
virus.
The original SFV expression system employs a plasmid in which the SP6
RNA polymerase promoter lies upstream of the cDNA version of the
SFV genome, modified such that the sP coding region has been replaced
by the gene of interest (lacZ in Fig. 1, B and
C). "Replicon" RNA (re-RNA) is transcribed and capped
in vitro, and transfected into mammalian cells where it is
amplified. Large amounts of target protein are generated from the
subgenomic message. Infectious particles carrying re-RNA can be
generated in vivo by cotransfection of "helper RNA" (5,
10). Helper RNA encodes the viral structural proteins found in the wild
type SFV, but lacks the packaging signal () found in the nsP2 open
reading frame. As a result, only re-RNA is packaged within a wild type viral coat, generating a recombinant virus that is capable of one round
of infection.
Several features of the SFV have made it a useful vector for the
expression of foreign genes. First, and in contrast to baculovirus, SFV
will infect almost any mammalian cell. Second, it is possible to
generate reasonable amounts of protein simply by transfecting re-RNA
(10). This approach avoids generating any virus; however, it is limited
by transfection efficiency and requires large amounts of RNA. Third,
virus can be produced quickly and efficiently in 2 days (10), which
makes it the fastest system available. Fourth, amplification by SFV
replicase is so efficient that the level of target protein can reach up
to 25% of total cellular protein (10). Finally, mutations within the
glycoprotein gene p62 (Fig. 1A) prevent infection unless the
virus is proteolytically treated with -chymotrypsin (11). This
safeguard feature significantly decreases the likelihood of generating
replication proficient virus (RPV) since two events would be required;
recombination between re-RNA and helper RNA, and reversion or
suppression of the conditional mutation. SFV vectors have been used to
generate several recombinant proteins (see, for example, Refs. 10, 12, and 13; reviewed in Ref. 14), to produce hybrid viruses (15, 16), and
to study aspects of the SFV life cycle.
The original SFV expression system (Fig. 1B) is hampered by the necessity to generate capped RNA transcripts in vitro and requires specialized conditions for RNA handling. To overcome these obstacles, we constructed a DNA-based self-amplifying SFV vector by replacing the SP6 promoter used in the original system with the RNA polymerase II-dependent cytomegalovirus immediate early (CMV IE) enhancer/promoter, which drives transcription in vivo (Fig. 1B). Transfection of this vector into BHK cells generated high levels of protein (20-30 pg/cell), production of which was dependent on a functional SFV polymerase. To complete the DNA-based expression system, a helper plasmid was constructed that encodes SFV sPs. Cotransfection of replicon and helper plasmids generated conditionally infectious virus capable of producing protein at the same high yield as virus generated by the RNA approach. No RPV was detected in several recombinant SFV virus preparations, a distinct advantage over a DNA-based Sindbis system reported previously (17).
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EXPERIMENTAL PROCEDURES |
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Plasmids--
pSCA was built in three stages. (i) The SV40
polyadenylation signal was amplified by polymerase chain reaction (PCR)
using SVhRB (18) as a template, Vent DNA polymerase, and the following primers: O-SV1 (SpeI):
5'-gccgactagtgatcataatcagccata-3' and O-SV2 (XbaI): 5'-gccgtctagatccagacatgata-3'. The
0.25-kb product was cut with SpeI and XbaI, and
ligated into SpeI-cut pSFV1 (10). The resulting plasmid,
pSFVpoly(A), contains a poly(A) signal downstream of the multiple
cloning site. (ii) The SP6 promoter and the first 513 base pairs of the
5' end of the SFV genome were removed from pSFVpoly(A) by digestion
with SphI and BsiWI (Fig. 1C). The
10.3-kb fragment was ligated with a 0.7-kb
SphI-EcoRI CMV-T7 fragment (generated by
amplification and digestion of base pairs 1570-2186 of pcDNA/NEO
(Invitrogen) with the primers O-CMV1 (SphI):
5'-ccgccggcatgcgtaatcaattacggggtc-3' and O-CMV2
(EcoRI): 5'-gccggaattcaagcttccggtctcccta-3'),
and a 0.57-kb EcoRI-BsiWI fragment containing the
first part of the SFV genome (generated by amplification and digestion
of base pairs 1-517 in pSFV1 with the primers
O-SFV3(EcoRI):
5'-gccggaattcatggcggatgtgtgacat-3' and reverse primer,
O-SFV2: 5'-gtacagcgatgttggtgc-3'). The resulting plasmid, pSCA1,
contains the CMV IE/T7 promoters upstream of the SFV replicon and
poly(A) region. (iii) pSCA
was built by ligating a 4.6-kb
BglII-SpeI fragment from pSFV3-lacZ (5, 10) to a 9.9-kb BglII-SpeI pSCA1 fragment. This places the
lacZ gene immediately downstream of the nsP4 coding region,
under the control of the SFV subgenomic promoter (Fig. 1C).
pSCA
was built by deleting a 2.7-kb SacII fragment
from pSCA
. pSCAHelper (Fig. 6A), was constructed by
inserting the 3.6-kb SpeI/AccI fragment from
pSCA1 into the 5.1-kb AccI/SpeI fragment from
pSFVHelper2 (5, 11).
Cell Culture Materials-- BHK-21 baby hamster kidney cells and COS-1 cells (SV40 transformed Green monkey kidney) were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal bovine serum (FBS), 25 mM HEPES buffer, and L-glutamine. Cells were incubated in a humidified atmosphere of 5% CO2 in air.
Electroporation--
BHK-21 and COS-1 cells were grown to 50%
confluence, trypsinized, washed once with IMDM plus FBS, once with PBS
and resuspended at 107 cells/ml in a sterile medium
comprising IMDM with 10 mM glucose and 0.1 mM
-mercaptoethanol. At room temperature, cells (0.8 ml) were
transferred to a 0.4-cm cuvette (Bio-Rad), DNA added, and
electroporation carried out using the Bio-Rad Gene Pulser. Cells from
one cuvette were plated on five 6-cm plates. To optimize the
transfection efficiency, variations in capacitance (25-960 mF),
voltage (0.1-0.4 kV), number of pulses (1 or 2), and delay between
pulses (0 or 30 s) were carried out in 80 combinations. In initial
experiments, two 30-s delayed pulses at 0.4 kV and 960 mF yielded the
maximum lacZ expression. However, re-optimization was
required, and in more recent experiments cells received one pulse at
0.4 kV and 960 µF. Each data point represents the average of three
electroporations.
-Galactosidase Assay--
-Galactosidase activity was
measured by an ONPG
(O-nitrophenyl-
-D-galactopyranoside,
Ampresco) assay as described previously (19).
-Galactosidase
standards (Boehringer Mannheim) were used to convert the readings of
A420 nm for the ONPG assay results into
nanograms of protein.
X-Gal Assay--
Transfected BHK-21 and COS-1 cells were stained
by the X-gal (5-bromo-4-chloro-3-indolyl--D-galactoside,
Chemica Alta Ltd.) method (20).
Southern Blot Analysis--
BHK-21 cells (8 × 106), electroporated with 2 µg of plasmid, were split
into five 6-cm dishes (i.e. 0.4 µg of DNA/plate). Total DNA from one plate was isolated by phenol-chloroform extraction (19),
and 15% of the sample was digested and run on a 0.8% agarose gel, and
transferred to a Gene Screen Plus membrane (NEN Life Science Products)
as described previously (21). The 3.1-kb BamHI fragment of
pSCA, corresponding to the lacZ gene, was random-prime labeled (22) with [
-32P]dCTP and used as a probe for
hybridization. Hybridization was performed as described (21). Blots
were washed twice in 2× SSC and 1% SDS, then twice again in 0.1× SSC
and 0.1% SDS at 65 °C. Signal was visualized and quantitated using
a Molecular Diagnostics PhosphorImager.
Generation of Infectious SFV Particles--
The transfected
amount of pSCA and pSCAHelper DNA was optimized to generate the
highest viral titer. In duplicate experiments, pSCA
(2 µg) and
pSCAHelper were added in molar ratios of 1:0.5, 1:1, 1:2, and 1:4,
respectively, to electroporation media containing 8 × 106 BHK-21 cells. Cells were electroporated and the
contents of each cuvette plated on a 10-cm tissue culture plate. After
4 h, the medium was aspirated, cells washed with PBS, and 8 ml of
IMDM (plus 10% FBS) was added. Medium containing the SFV particles was
collected 24 and 36 h after electroporation and clarified by
centrifugation at 2000 rpm (Sorvall RT 6000 D) for 15 min at 4 °C.
Virus was activated with 0.5 mg/ml
-chymotrypsin at room temperature
for 45 min, and aprotinin (0.38 mg/ml) added to stop protease activity
(11). 0.2 ml of activated virus stock was added to 106
BHK-21 cells on 6-cm tissue culture plates, and incubated for 45 min at
37 °C in a humidified atmosphere of 5% CO2 in air. The infectious medium was aspirated, cells washed with PBS, and IMDM/10% FBS was added. ONPG and X-gal assays were performed 18 h after infection. X-gal-positive and -negative cells were counted in 10 fields
viewed under 400× magnification. Total number of cells on the dish was
obtained by trypsinizing a duplicate plate of infected cells. Viral
titer was estimated by multiplying the total number of cells by both
the fraction of X-gal-positive cells and a factor to correct for the
volume of virus-containing supernatant used in the assay.
Determination of Replication Proficient Virus Particles--
A
total of 8 × 106 BHK-21 cells were transfected with a
1:1 molar ratio of pSCA (2 µg) and pSCAHelper (1.2 µg) in 10 separate reactions. At 24 h after transfection, 0.5 ml of the
growth medium from each 10-cm tissue culture plate was collected and
viral titer determined (see above). Without
-chymotrypsin treatment,
the remaining 6 ml were divided among six tissue culture plates (6 cm)
containing 1 × 106 BHK cells and incubated for 45 min. The virus-containing medium was aspirated, and the cells were
washed in PBS and then overlaid with a plaque assay medium (PAM)
containing 0.6% low melt agarose (Bio Shop, Canada) in IMDM (plus 2%
FBS, 10% tryptose phosphate (Life Technologies, Inc.), and 20 mM MgCl2). The cells were incubated for 5 days
and checked for plaques daily.
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RESULTS |
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Reporter Gene Expression in Vitro--
The current expression
system based on SFV requires transfection of RNA that contains the
viral replicase genes and the inserted gene of interest (Fig.
1B). RNA is obtained by
SP6-driven in vitro transcription of an SFV cDNA-geneX
cassette. Our goal was to simplify this system by circumventing the
need for in vitro transcription and RNA handling. Thus, two
major modifications were made to the parent vector pSFV1 (5, 10).
First, the SV40 termination/polyadenylation signal was added
immediately downstream of the 69 A residues at the 3' end of the SFV
cDNA (23). The message expressed from the final vector is expected
to have two poly(A) stretches separated by a short SV40 sequence.
Second, the SP6 promoter in pSFV1 was replaced by a hybrid CMV IE/T7
promoter derived from the pcDNA vector (Invitrogen). The final
vector, pSCA, was completed by insertion of the reporter gene,
lacZ, into the multiple cloning site (Fig. 1). This
bifunctional vector can be transcribed in vivo or in
vitro using either the CMV IE or T7 promoter, respectively.
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pSCA Expresses More
-Galactosidase than CMV
Gal--
The
levels of
-galactosidase obtained with the pSCA
vector should be
much higher than those obtained from a comparable vector that lacks the
SFV nsPs. Message levels from CMV
Gal (CLONTECH) are dependent on CMV IE promoter activity alone. Thus, BHK-21 cells
were transfected with pSCA
and CMV
Gal and
-galactosidase activity measured over 5 days after transfection (Fig.
3A). The amount of
-galactosidase produced by pSCA
reached a maximum on day 2, and
on day 1 for the CMV
Gal vector. In several replicate experiments,
the expression levels obtained with pSCA
were, on average,
20-30-fold greater than CMV
Gal. Similar data were obtained using
COS-1 cells (data not shown).
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Reporter Gene Expression from pSCA Is Dependent on Functional
Replicase Proteins--
To confirm that the SFV nsPs were indeed
essential to the activity of pSCA
, we tested whether a derivative
with a large deletion in nsP2-3 could direct
-galactosidase
expression. This plasmid (
pSCA
, Fig. 1C) was
transfected into BHK-21 cells and
-galactosidase activity measured
over a 5-day period. pSCA
expressed an average of 220 ng of
-galactosidase/6-cm plate, but there was no
-galactosidase activity in cells transfected with
pSCA
(Fig.
4A). Similarly, when BHK-21
cells were stained for
-galactosidase using X-gal, only
pSCA
-transfected cells were positive (Fig. 4B). Southern analysis performed on cells taken from 2, 3, and 4 days after transfection confirmed that both pSCA
and
pSCA
plasmids had been taken up (Fig. 4C). The DNA from each transfection was
quantitated on a PhosphorImager and the data used to base-line correct
the
-galactosidase levels (Fig. 4A). Thus,
-galactosidase expression from pSCA
is not due to read-through
translation of the full-length message, but is dependent on the SFV
polymerase, which carries out both amplification and synthesis of the
subgenomic lacZ transcript (Fig. 1A).
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pSCA Expresses More Protein than a DNA-based Sindbis
Vector--
Sindbis, another alphavirus, was engineered by Wolff and
co-workers (24) into a plasmid-based vector. In this system, in vivo transcription of the Sindbis RNA was achieved by placing viral cDNA under transcriptional control of the Rous sarcoma virus long terminal repeat (RSV LTR). The Sindbis vector, pSin-nlLacZ, also
utilized lacZ as a reporter gene. To compare expression from the SFV (pSCA
) and Sindbis (pSin-nlLacZ) DNA-based vectors, pSCA
and pSin-nlLacZ were transfected into BHK-21 cells and
-galactosidase activity was measured over a 5-day period (Fig.
5A).
-Galactosidase expression peaked on day 2 for both pSCA
and pSin-nlLacZ. In several
replicate experiments,
-galactosidase expression in BHK-21 cells was
10-20-fold higher for pSCA
than for pSin-nlLacZ. Transfection efficiency of both plasmids, as assessed by Southern analysis, was
similar (data not shown). Thus, higher levels of
-galactosidase were
obtained using the SFV vector.
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Generation of Infectious Recombinant SFV Using DNA-based
Vectors--
To generate infectious virus particles using DNA-based
SFV vectors, we constructed pSCAHelper, in which expression of helper RNA is driven by the CMV IE promoter (Fig.
6A). This RNA contains the
sequences at each end of the SFV genome that mediate amplification by
the nsPs, but lacks the packaging signal located in the nsP2 gene (Fig.
1A). Thus, cotransfection with the replicon vector will
result in amplification of the helper message, transcription of the
subgenomic message, and translation of the sPs. The sPs will package
re-RNA, but not helper RNA. pSCAHelper carries mutations in the p62 sP
gene that alter three amino acids so that generation of E2 and E3
glycoproteins from the p62 precursor and activation of the virus
requires -chymotrypsin treatment (11). BHK-21 cells were transfected
with pSCA
and pSCA-Helper plasmids in four molar ratios. After
24 h, 0.2 ml of the growth medium from the transfected cells was
activated with
-chymotrypsin, applied to fresh BHK-21 cells, and
titer determined. The highest viral yield was obtained with a 1:1 molar
ratio, followed by 1:2, 1:0.5, and 1:4 (Fig. 6B).
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Absence of Replication Proficient Viruses-- One of the problems associated with any viral expression system is the generation of wild type virus through recombination between the separated components of the viral genome. In alphavirus systems, replication proficient virus (RPV) particles have been detected following cotransfection of replicon and helper RNAs (11, 25). Recombination occurs when the polymerase switches templates during amplification (26). In a DNA-based system, homologous recombination between plasmids provides another level at which a complete virus genome could be generated. However, with the SFV system described here, recombination alone would not generate fully infectious virus since reversion or suppression of the conditional mutation that inhibits p62 cleavage would also be required (11). We performed three assays to estimate the frequency of RPV generation with the DNA-based SFV system (Fig. 7).
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DISCUSSION |
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Alphavirus expression systems have been used to express a wide range of heterologous proteins (reviewed in Refs. 14 and 27). Although the approach is simpler and much faster than vectors such as baculovirus, neither SFV nor the related Sindbis vectors have superceded baculovirus as the expression system of choice. One technical drawback of the original alphavirus systems is that they require production and manipulation of recombinant RNA in vitro. Therefore, the design of DNA-based alphavirus vectors, both for SFV (this work) and Sindbis (17, 24), significantly improves the utility of these expression systems. These developments also simplify the use of alphaviruses as in vivo gene delivery systems, for example, in the delivery of vaccines (28).
Our observations, and those of others using DNA-based Sindbis vectors
(17, 24), show that at least a portion of full-length re-RNA generated
in the nucleus must reach the cytoplasm. Indeed, the amount of protein
produced per cell with the DNA-based SFV vector was similar to that
obtained previously following infection of cells with a recombinant
virus (10). Expression was optimal with 2 µg of pSCA and peaked
48 h after transfection. With these parameters, approximately
20-30 pg of
-galactosidase was obtained per cell. Protein
expression with the DNA-based SFV vector was 20-30-fold higher than
with a simple CMV IE-based vector. This difference can be attributed to
the effect of the SFV polymerase, since a large deletion in the nsP
gene region completely abolished protein expression.
Two groups have developed DNA-based Sindbis expression vectors (17,
24). We compared the performance of the Sindbis vector pSin-nlLacZ (24)
against the SFV vector, pSCA. pSCA
generated 10-20-fold more
-galactosidase than the Sindbis-derived vector. This result is not
due to a difference in the efficiency of SFV and Sindbis polymerases,
since transfection of SFV or Sindbis re-RNAs yields similar levels of
protein (10, 27). Thus, other components of pSCA
and pSin-nlLacZ
must be responsible. pSin-nlLacZ encodes
-galactosidase that is
targeted to the nucleus, whereas the protein encoded by pSCA
lacks a
nuclear localization signal, but it seems unlikely that this would
explain the large difference in expression levels. An important element
required for maximal expression is the viral poly(A) tract placed
downstream of the reporter gene (17). However, both pSCA
and
pSin-nlLacZ contain such a motif. pSCA
contains an additional
termination/polyadenylation signal downstream of this tract, whereas
pSin-nlLacZ lacks this element. However, this motif inhibited
expression of another DNA-based Sindbis vector (24). Other factors that
could influence protein expression include the promoters (CMV IE or RSV
LTR in pSCA
and pSin-nlLacZ, respectively), the positioning of
lacZ relative to the subgenomic promoter, and the efficiency
of translation. We suspect that promoter-strength is the major factor,
but additional experiments are required to test this idea. Studies with
other DNA-based Sindbis vectors also suggest that promoter-strength influences vector efficiency (17).
We also utilized the CMV IE promoter to generate a helper plasmid
(pSCAHelper) in which expression of viral sPs is RNA polymerase II-dependent. Cotransfection of pSCA and pSCAHelper
generated virus at a titer of around 106 infectious
particles/ml (which could be artificially low, since X-gal staining was
used to determine titer, and this method has recently been shown to
underestimate the number of
-galactosidase-positive cells (20)).
Virus generated using the new DNA vectors gives the same high yield of
protein as virus generated by the original RNA-based approach (50 µg
of
-galactosidase/106 particles of recombinant
virus).
Virus stocks generated using pSCAHelper are conditionally infectious
due to amino acid changes within sP p62 at the junction between the E2
and E3 spike proteins (11). This modification reduces the chance of
obtaining wild-type virus, which would require recombination between
re-RNA and helper RNA, and reversion or suppression of the conditional
mutation. Empirical measurements show that the chance of generating
RPV, following transfection of re-RNA and helper RNA that encodes
-chymotrypsin-activated p62, is less than 10
10, and
has never been observed (11). A disadvantage of DNA vectors is that
they provide another opportunity for recombination. The level of RPVs
obtained following transfection of Sindbis DNA-based replicon and
helper vectors was 1000-fold higher (~107 PFU/ml) than
that obtained after transfection of in vitro generated re-RNA and helper RNA (~104 PFU/ml) (17). Indeed, a
similar number of RPVs and recombinant particles were generated using
the DNA-based system Sindbis vectors. In contrast, no RPVs were
detected in a population of nearly 108 recombinant
particles generated using the DNA-based SFV system described here.
These statistics highlight an important advantage of the SFV
vectors.
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ACKNOWLEDGEMENTS |
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We thank Shi-Yen Mak for help in building
pSCA and
pSCA
, and Irina Burcescu for help in building
pSCAHelper. We are also very grateful to P. Liljeström for
providing pSFV1, pSFVHelper, and pSFV3-lacZ, and H. Herweijer and
J. A. Wolff for providing pSin-nlLacZ.
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FOOTNOTES |
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* This work was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society, the Virginia S. Boyce Research Fund of Prevent Blindness America, and the RP Research Foundation - Fighting Blindness.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a Medical Research Council of Canada studentship.
** To whom correspondence should be addressed: Eye Research Institute of Canada,399 Bathurst St., Toronto, Ontario M5T 2S8, Canada. Tel.: 416-603-5865; Fax: 416-603-5126; E-mail: rbremner{at}playfair.utoronto.ca.
1
The abbreviations used are: SFV, Semliki Forest
virus; nsP, non-structural protein; sP, structural protein; re-RNA,
replicon RNA; RPV, replication proficient virus; CMV IE,
cytomegalovirus immediate early; X-gal,
5-bromo-4-chloro-3-indolyl--D-galactoside; PAM, plaque
assay medium; PFU, plaque-forming unit; RSV LTR, Rous sarcoma virus
long terminal repeat; m.o.i., multiplicity of infection; BHK, baby
hamster kidney; PBS, phosphate-buffered saline; FBS, fetal bovine
serum; F, farad(s); kb, kilobase pair(s); IMDM, Iscove's modified
Dulbecco's medium; ONPG,
O-nitrophenyl-
-D-galactopyranoside.
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REFERENCES |
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