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Construction and Propagation of Human Adenovirus Vectors
|Construction and Propagation of
Human Adenovirus Vectors
Adenoviruses (Ads), which have been used extensively
as a model system for molecular studies of
mammalian cell DNA replication, transcription, and
RNA processing, are now being increasingly investigated
as potential mammalian expression vectors for
gene therapy and for recombinant vaccines (Berkner,
1988; Graham and Prevec, 1992; Hitt et al.
, 1999). There
are many reasons for this renewed popularity of Ad
vectors: the 36,000-bp double-stranded DNA genome
of Ad is relatively easy to manipulate by recombinant
DNA techniques; Ad infects a wide variety of mammalian
cell types, both proliferating and quiescent,
with high efficiency; the genome does not undergo
rearrangement at a high rate; the viral particle is relatively
stable; and the virus replicates to high titer
in permissive cells, producing up to 10,000 plaqueforming
units (PFU) per infected cell. Late in infection,
most of the infected cell protein is virally encoded,
potentiating the use of replication-proficient recombinant
Ads as short-term high-level expression vectors.
In nondividing, nonpermissive cells the viral genome
may persist as an episome and continue to express for
long periods in vitro
. This holds true in vivo
as well in
the absence of an immune response against vectorinfected
cells. This article describes methods for inserting
foreign genes into the Ad genome and for
purifying, growing, and titrating the recombinant
viruses. Our vectors are based on the human Ad5
genome, the structure of which is shown in Fig. 1. In a
wild-type infection, early genes (Ela, Elb, E2, E3, and
E4) are expressed prior to DNA replication, and late
gene expression, driven predominantly by the major
late promoter at 16 map units, occurs after the initiation of DNA replication. Deletion of the E1 region
renders the virus replication defective, which is desirable
for most gene therapy applications. However,
such vectors must then be propagated in El-complementing
cells, such as the 293 cell line (Graham et al.
1977). Deletion of E3, which is nonessential for virus
growth in vitro
, together with deletion of El, allows
insertion of foreign genes up to about 8 kb in length.
Without deleting E3, the insertion capacity of Ad is
about 5 kb.
|FIGURE 1 Transcription map of the human adenovirus type 5. The
approximately 36-kb genome of Ad5 is divided here into 100 map
units. Messages from the early regions are indicated as light lines
and late messages are indicated in bold. Late transcription originating
from the major late promoter at 16 map units and terminating
near the right end of the genome is indicated by the open arrow. This
transcript is processed into five families of late mRNAs spliced to a
common tripartite leader (1, 2, and 3 at map units 16.5, 19.5, and
26.5, respectively), although some mRNA species contain additional
leaders. (For more details, see Ginsberg, 1984.)
The vector systems described here rely on site-specific
recombination in 293 cells between a shuttle
plasmid derived from the E1 region at the "left" end
of Ad and a larger plasmid carrying nearly the entire
Ad genome in a circular form (Fig. 2). In the Cre/loxP
system (Ng et al.
, 2000a), the genomic plasmid pBHGloxAE1,
E3Cre carries an expression cassette encoding
the Cre recombinase in a region of the plasmid that is
excluded from the final vector genome. Cre mediates
recombination between a loxP site downstream of
the E1 insertion site in the shuttle plasmid and a
loxP site in the E1 region of the genomic plasmid. The
FLP/frt system (Ng et al.
, 2000b) is identical except
that it uses the FLP recombinase to mediate recombination
between frt sites in the shuttle and genomic
plasmids. The Ad packaging signal has been deleted
from both genomic plasmids, which virtually eliminates
the generation of nonrecombinant infectious
progeny following cotransfection with an E1 shuttle
|FIGURE 2 Construction of Ad vectors by two-plasmid site-specific recombination in 293 cells. The AdMax strategy
used to introduce foreign DNA inserts into the E1 and/or E3 regions for rescue into virus is illustrated.
Expression cassettes can be inserted in place of the E1 region (Ad5 nucleotides 455-3523) by cloning into E1
shuttle plasmids carrying the recognition site (e.g., loxP) for a site-specific recombinase. E1 shuttle plasmids
are described further in Fig. 3. The 293 cells are then cotransfected with this shuttle plasmid and an Ad
genomic plasmid carrying the appropriate recombinase (Cre for loxP-containing shuttles or FLP for frtcontaining
shuttles). Genomic plasmids are available with E3 deleted (Ad5 nucleotides 28138-30818;
pBHGloxΔE1,E3Cre or pBHGfrtΔE1,E3FLP) as shown or with a wild-type E3 region (pBHGloxE3Cre and
pBHGfrtE3FLP) (Ng and Graham, 2002). Expression of recombinase in 293 cells results in recombination
between recognition sites in the shuttle and in the genomic plasmid, generating an E1 replacement Ad vector
(top vector in illustration). E3 replacement Ad vectors are constructed by inserting the transgene expression
cassette into a unique PacI site that replaces the E3 region in the Ad genomic plasmid. The 293 cells cotransfected
with this genomic construct and an "empty" (i.e., no transgene) E1 shuttle plasmid will produce a
replication-defective E3 replacement Ad vector (middle vector in illustration). Note that it is also possible to
cotransfect with a plasmid containing the intact left end of Ad5 to produce an E1+ nondefective vector by
overlap recombination (Bett et aI., 1994). Double recombinant vectors are generated by recombination between
an E1 shuttle plasmid carrying one expression cassette and a genomic plasmid carrying a second expression
cassette (bottom vector in illustration). Ad and bacterial sequences are indicated by thick and thin black bars,
respectively, loxP and frt sites by open triangles, inverted terminal repeats (ITRs) by black arrows, and the
packaging signal by the symbol Ψ.
With either system, vectors can be generated with
inserts in place of El, E3, or both (Fig. 2). E1 replacement
vectors, by far the most common, are constructed
by insertion of a transgene expression cassette into the
E1 shuttle plasmid and subsequent rescue by recombination
with the genomic plasmid in 293 cells. Some of the most commonly used shuttle plasmids for this
system are illustrated in Fig. 3. E3 replacement vectors
are constructed by inserting the transgene expression
cassette directly into the genomic plasmid at the PacI
site engineered in place of E3 (Bett et al.
, 1994). The
genomic plasmid carrying an insert in E3 can be
cotransfected with a shuttle plasmid carrying no insert
(i.e., E1 deleted) to generate a replication-defective
vector. Double recombinant Ad vectors can be produced
by cotransfecting 293 cells with the E3 replacement
genome-size plasmid together with an E1 shuttle
plasmid encoding a second transgene. This latter strategy
has been used successfully to rescue a recombinant
Ad vector containing the p35 subunit of interleukin-12
(IL-12) in E1 and the IL-12 p40 subunit in E3 (Bramson et al.
|FIGURE 3 Structure of E1 shuttle plasmids used for vector rescue by in vivo site-specific recombination. The
shuttle plasmids pDC311, pDC312, pDC315, and pDC316 are used to rescue vectors by Cre-mediated recombination.
The shuttle plasmids pDC511, pDC512, pDC515, and pDC516 are used to rescue vectors by FLPmediated
recombination. Plasmids pDC311, pDC312, pDC511, and pDC512 are designed for insertion of a
cassette consisting of a promoter, transgene, and polyadenylation signal sequence. The polycloning sites of
plasmids pDC315, pDC316, pDC515, and pDC516 are flanked 5' by the murine CMV promoter and 3' by the
SV40 polyadenylation signal sequence. Coding sequences cloned into the latter plasmids generate vectors
with high levels of expression in both human and murine cells (Addison et al., 1997).
Foreign coding sequences, including their own or
heterologous promoters, can be inserted into the
shuttle plasmids or into the E3 region of the pBHG
plasmids in an orientation either parallel or antiparallel
to the E1 or E3 transcription unit. In general, higher
expression levels have been obtained with inserts in
the parallel orientation in either E1 or E3 (unpublished
results); however, the sequence of the insert itself can
affect expression levels, particularly for E3 insertions.
Once the desired plasmids have been constructed, the
following protocols are used to produce and purify the
recombinant Ad viruses.
II. MATERIALS AND
The Cre/loxP and FLP/frt based AdMax vector
rescue systems are available, as Kit D (Cat. No. PD-01-
64) and Kit E (Cat. No. PD-01-65) respectively, from
Microbix Biosystems, Incorporated. Minimal essential
medium (MEM) Fll (Cat. No. 61100-087), L
(Cat. No. 25030-081), penicillin/streptomycin (Cat. No.
15140-122), horse serum (Cat. No. 16050-159), newborn
calf serum (NCS) (Cat. No. 16010-159), agarose (Cat.
No. 15510-027), and dithiothreitol (Cat. No. 15508-013)
can be obtained from Invitrogen. Bovine serum
albumin fraction V (Cat. No. A2153), fetal bovine
serum (FBS) (Cat. No. F4135), Joklik's modified MEM
(Cat. No. M0518), salmon sperm DNA (Cat. No.
D1626), and orcein (Cat. No. 07380) are available from
Sigma Chemical Company. All sera are inactivated
prior to use by heating to 56°C for 30min. Fungizone
can be purchased from Bristol-Myers-Squibb (Cat. No.
043780). Nunc tissue culture dishes (Cat. No. 1-
68381A) can be obtained from VWR. Sterile petri
dishes (Cat. No. 08-757-12), Difco agar (Cat. No. 0145-
17-0), Difco Bacto Lennox LB broth base (Cat. No. 0402-
07-0), Becton Dickinson BBL trypticase peptone (Cat.
No. Bl1921), and yeast extract (Cat. No. Bl1929) can be
obtained from Fisher Scientific. Pronase (Cat. No.
1459643) and bovine pancreatic deoxyribonuclease I
(Cat. No. 104-159) can be purchased from Roche Diagnostics.
Analytical grade CaCl2·
O (Cat. No. B10070)
can be obtained from BDH. All other chemicals can be
purchased from standard chemical suppliers (e.g.,
BDH). Spinner flasks (1969 series) can be obtained
from Bellco and Pierce Slide-A-Lyzer 10K dialysis cassettes
(Cat. No. 66425) from Chromatographic Specialities.
Beckman SW41 Ti and SW 50.1 rotors are also
required. Reagents for plasmid DNA isolation, as well
as restriction enzymes and reagents and apparatus for
horizontal slab gel electrophoresis, are described in a
number of cloning manuals (e.g., Sambrook and
A. Preparation of Plasmid DNA for
In order to minimize the generation of bacterial
clones containing rearranged plasmid DNA, which
is occasionally observed in preparations of very
large plasmids such as pBHGloxΔE1,E3Cre and
pBHGfrtΔE1,E3FLP, we have adopted the following protocol for bacterial growth prior to plasmid DNA
- Super broth (SB): Dissolve 5g NaCl, 32 g trypticase
peptone, 20 g yeast extract, and 1 g glucose in 1 liter
H2O. Add 5 ml 1 N NaOH. Sterilize by autoclaving.
- LB-agar plates: Dissolve 10g BBL Lennox LB broth
base in 500ml H2O. Add 7.5g agar and sterilize by
autoclaving. Cool LB-agar to about 50°C, add
antibiotics as required, and pour 25 ml into each of
20 sterile petri dishes. Store at 4°C.
- Reagents for isolating plasmid DNA on CsCl gradients:
Not described here.
B. DNA Transfection for Rescue of
Recombinant Adenovirus Vectors: Calcium
- Streak plasmid-bearing bacteria on an LB-agar
plate containing appropriate antibiotics and grow
overnight at 37 °C.
- Pick two or more colonies off the plate, resuspend
each in 5 ml SB plus antibiotics, and incubate at
37 °C on a shaker for several hours.
- Add each 5ml culture to 500ml SB plus antibiotics
and continue incubating overnight.
- Purify the plasmid DNA from each culture separately
by alkaline lysis of the bacteria and CsCl
banding as described in standard cloning manuals
(e.g., Sambrook and Russell, 2001). Plasmid DNA that
has not undergone any detectable rearrangement, as
indicated from comparison between predicted and
observed restriction enzyme cleavage pattens, is suitable
for use in cotransfections.
- Complete MEMF11 (or Joklik's modified MEM): Add
5 ml 0.2M L-glutamine, 5 ml penicillin/streptomycin
(10,000U/ml and 10mg/ml, respectively), and 5ml
0.25mg/ml fungizone to 500ml MEMF11 (or Joklik's
modified MEM). Store at 4°C for up to 2 weeks. Add
55 ml heat-inactivated NBS, FBS or HS prior to use in
- 10X citric saline: Dissolve 50g KCl and 22 g trisodium
citrate dihydrate (Na3C6H507.2H2O) in H2O to a final volume of 500ml. Sterilize by autoclaving and
store at 4°C. Dilute 1:10 in sterile H2O to prepare 1X
- HEPES-buffered saline (HEBS): Dissolve 5g
HEPES free acid, 8g NaCl, 0.37g KCl, 0.1 g Na2HPO4,
and 1 g glucose in 900ml H2O. Adjust pH to 7.1. Adjust
volume to 1 liter with H2O. Aliquot into small glass
bottles, sterilize by autoclaving, and store at 4°C.
- 10X SSC: Dissolve 8.7 g NaCl and 4.4 g tri-sodium
citrate dihydrate in H2O to a final volume of 100ml.
Adjust pH to 7.0. Autoclave. Prepare 0.1X SSC by diluting
10X SSC and then autoclaving.
- 2mg/ml carrier DNA: Dissolve 100mg salmon
sperm DNA in 50ml sterile 0.1X SSC by stirring
overnight at room temperature. Determine concentration
by reading the OD at 260nm (one OD unit =
50µg/ml). Store in small aliquots at -20°C.
- 2.5M CaCl2: Add H2O to 36.8 g CaCl2.2H2O (analytical
grade) to a final volume of 100ml. Sterilize by
filtration and store in small plastic tubes at 4°C.
- MEMF11-agarose overlay: To make approximately
200ml, add 10ml horse serum (HS) and 2ml each of Lglutamine,
penicillin/streptomycin, fungizone (at concentrations
given earlier), and autoclaved 5% yeast
extract (w/v in H2O) to 100ml 2X MEMF11. Autoclave
1 g agarose in 100ml H2O. Bring the 2X medium and
agarose to 44°C before mixing and use within an hour.
C. Screening Adenovirus Plaque Isolates
- Grow monolayer cultures of 293 cells in 150-mm
dishes in complete MEMFll medium plus 10% FBS (or
NBS). At 90% confluence, remove medium, wash each
dish twice with 10ml 1X citric saline, and then incubate
for a maximum of 15 min at room temperature in
3 ml 1X citric saline to detach cells. Resuspend cells in
medium and divide between two or three 150-ram
dishes. 293 cells should be refed with fresh medium
every 3 days if not ready to passage.
- Set up low-passage (<p40) 293 cells in 60-mm
dishes to be about 70-80% confluent at the time of use.
As a rule of thumb, one 150-mm dish of nearly confluent
293 cells can be split into eight 60-mm dishes
each containing 5ml complete MEMFll + 10% FBS,
which will be ready for transfection the next day.
- Add 0.005 volume 2mg/ml carrier DNA to 1X
HEBS and shear by vortexing for 1 min.
- For each virus to be rescued, aliquot 2ml HEBS
+ carrier DNA (enough for four dishes) into each of
three sterile clear plastic tubes.
- To these tubes add E1 shuttle plasmid DNA
and the appropriate genomic plasmid (e.g., pBHGloxΔE1,
E3Cre) in the following amounts: 20µg of
each plasmid, 8µg of each plasmid, and 2µg of each
plasmid. If a negative control is desired, set up similar coprecipitations omitting the first plasmid. A useful
positive control is 2µg of the infectious plasmid
- Gently mix by shaking and then slowly add
0.1 ml 2.5M CaCl2 to each tube.
- Gently mix and let stand at room temperature for
15-30 min. (A fine precipitate should form within a few
minutes.) Without removing the growth medium, add
0.5ml DNA suspension to each dish of cells (four
dishes for each tube of coprecipitate) and then incubate
at 37°C in a CO2 incubator for at least 5h, preferably
- Remove the medium and add to each dish 10ml
MEMF11-agarose overlay previously equilibrated to
44°C. After the agarose solidifies, incubate at 37°C. Plaques should appear after about 5-14 days. When
dishes are examined from below by eye, plaques
appear turbid as a consequence of light scattering by
dead cells in an otherwise smooth cell monolayer.
Microscopic examination reveals plaques as zones of
dead or lysed cells surrounded by rounded infected
- At about 10 days posttransfection, pick wellisolated
plaques by punching out agar plugs from the
cultures using a sterile Pasteur pipette. Transfer each
agar plug to 1 ml sterile PBS++ + 10% glycerol in a sterile
vial. Store at -70°C until use.
The following protocol describes the expansion of
plaque isolates by growth in monolayer cultures of 293
cells. A portion of the infected cell material is stored
for further purification; the remainder is harvested for
analysis of viral DNA.
- Phosphate-buffered saline++ (PBS++): To make solution
A, dissolve 80g NaCl, 2g KCl, 11.5g Na2HPO4,
and 2g KH2PO4 in H2O to a final volume of 1 liter.
To make solution B, add 1 g CaCl2.2H2O to 100 ml H2O.
To make solution C, add 1 g MgCl2.6H2O to 100ml
H2O. Sterilize solutions separately by autoclaving. For
100 ml PBS++ mix 88 ml sterile H2O with 10 ml solution
A and 1 ml each of solutions B and C.
- PBS++ + 10% glycerol: Add 10 ml sterile glycerol to
- Pronase stock solution: Dissolve 0.5g pronase in
100ml 10mM Tris-HCl, pH 7.5; heat at 56°C for 15 min
and then incubate at 37°C for 1 h. Aliquot and store at
-20°C To prepare working solution, thaw stock solution
just before use and add 0.1 volume to 10mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.5% (w/v) sodium
dodecyl sulfate (SDS).
- Complete MEMF11 + 5% HS: Add 25ml HS to
475 ml complete MEMF11.
- 0.1X SSC: See Section III.B, solution 4.
- Reagents for restriction analysis of viral DNA: Not
D. Plaque Assays for Purification and
Titration of Adenovirus
PBS++ and MEMF11-agarose overlay (at 44°C):
- Set up 60-mm dishes of 293 cells to be 80-90%
confluent at time of infection. The denser and older the
cell monolayer, the longer it takes for the cytopathic
effect to reach completion.
- Remove medium from 293 dishes and add 0.2 ml
virus (agar plug suspension). Rock dishes once and
adsorb at room temperature for 30 min. Add 5 ml complete
MEMF11 + 5% HS and incubate at 37°C
- A cytopathic effect should be visible within 1-2
days. Harvest virus and extract infected cell DNA
(steps 4 to 6) when all cells are rounded and most have
detached from the dish (usually 3-4 days).
- Release semiadherent cells from the dish by
gentle pipetting. Transfer 3.5 ml of the cell suspension
to a sterile vial containing 0.5 ml sterile glycerol. Store
at -70°C until you wish to amplify the vector.
- Transfer the remaining 1.5 ml to a microfuge tube
and spin 2min at 7000rpm. Aspirate all but about 0.1
ml supernatant. Vortex well to suspend infected cells.
To extract DNA, add 0.5 ml pronase working solution
to the cells in the microfuge tube and incubate at 37°C for 4-18 h.
- Add 1 ml cold 96% ethanol to precipitate the
DNA. Mix well by inverting the tube several times-a
fibrous precipitate should be easily visible and the
solution should no longer be viscous. Spin 5min at
14,000rpm and then aspirate supernatant. Wash pellet
twice with 70% ethanol and air dry.
- Dissolve DNA pellet in 50µl 0.1X SSC by heating
at 65°C with occasional vortexing. Digest 5µl with HindIII (1 unit overnight is usually sufficient for complete
- Apply digested samples and appropriate
markers (a HindIII digest of wild-type Ad5 being one
convenient marker) to a 1% agarose gel containing
ethidium bromide and subject to electrophoresis until
the dye front has migrated at least 10cm. If the cytopathic
effect was complete, viral DNA bands should be
easily visible (under ultraviolet light) above a background
smear of cellular DNA. Note that in HindIII digests of human DNA there will be a band of cellular
repetitive DNA at 1.8 kb.
- Verify candidate recombinants using other diagnostic
restriction enzymes. Although generally 100%
of viral plaques obtained using AdMax are correct, it
is good laboratory practice to carry out one round of
plaque purification, as described later, and screening as described in thissection prior to preparation of
E. Preparation of High-Titer Viral Stocks
(Crude Lysates) from Cells in Monolayer
- Set up 60-mm dishes of 293 ceils to be confluent
at time of infection.
- Remove medium from dishes. Add 0.2ml virus
(dilution of agar plug suspension in PBS++ if you wish
to plaque purify or dilution of stock for titration). We
typically assay dilutions ranging from 10-3 to 10-6 for
plaque purification or 10-4 to 10-10 for virus titration.
Adsorb the virus for 30-60min in an incubator,
occasionally rocking the dishes. Add 10ml MEMF11-
agarose overlay, cool, and then continue incubation at
- Plaques should be visible within 4-5 days and
should be counted for titration at 7 days and again at
10 days. For plaque purification, proceed as for isolation
of plaques following transfections (Section III.C).
Because most of the virus remains associated with
the infected cells until very late in infection, high-titer
stocks can be prepared easily by concentrating infected
293 cells as described here.
PBS++, PBS++ + 10% glycerol, and complete MEMF11 +
: See Section III.C.
F. Preparation of High-Titer Viral Stocks
(Purified) from Cells in Suspension
- Set up 150-mm dishes of 293 cells to be 80-90%
confluent at time of infection. We generally use eight
or more dishes for each virus.
- To prepare high-titer stocks, remove medium
from the 293 cells and infect at a multiplicity of infection
(MOI) of 1-10 PFU per cell (1 ml diluted virus per
150-mm dish). For the initial stock preparation, we
dilute virus (from the untitered 4-ml sample stored at
-70°C after the last round of viral screening) 1:8 with
PBS++. To minimize generation and amplification of
rearranged forms of the vector, always prepare hightiter
stocks from viral screening samples, not from
CsCl-banded stocks (Section III.F).
- Adsorb for 30-60 min and then refeed with complete
MEMF11 + 5% HS. Incubate at 37°C and examine
daily for signs of a cytopathic effect.
- When the cytopathic effect is nearly complete,
i.e., most cells rounded but not yet detached, harvest by
scraping the cells off the dish, combining the cells plus
spent medium, and centrifuging at 800g for 15min.
Aspirate the medium and resuspend the cell pellet in
2ml PBS++ + 10% glycerol per 150-mm dish. Freeze
(-70°C) and thaw (37°C) the crude virus stock two or
three times prior to titration. Store aliquots at -70°C.
Recombinant Ads can be purified from crude
lysates of either monolayer or suspension cultures.
Due to the greater ease of handling suspension cultures,
however, this source is preferable for the preparation
of purified high-titer viral stocks as described
here. Similar yields can be obtained from thirty to sixty
150-mm dishes of 293 cell monolayers.
- Complete Joklik's modified MEM + 10% HS: Add
50ml HS to 450ml complete Joklik's modified MEM
(described in Section III.B). Store at 4°C.
- 1% sodium citrate: Dissolve 1 g trisodium citrate
dihydrate in H2O to a final volume of 100ml.
- Carnoy's fixative: Add 25 ml glacial acetic acid to
75 ml methanol.
- Orcein solution: Add 1 g orcein dye to 25 ml glacial
acetic acid plus 25ml H2O. Filter through Whatman
No. 1 paper.
- 0.1M Tris-HCl, pH 8.0: Add 1.2g Tris base to
80ml H2O. Adjust pH to 8.0 with HCl. Adjust volume
to 100 ml and autoclave.
- 5% Na deoxycholate: Add 5g Na deoxycholate to
100 ml H2O.
- 2 M MgCl2: Add 40.6 g MgCl2·6H2O to 100 ml H2O and sterile filter.
- DNase I solution: Dissolve 100mg bovine pancreatic
deoxyribonuclease I (DNase I) in 10ml of 10mM Tris-HCl, pH 7.4, 50mM NaCl, 1 mM dithiothreitol,
0.1 mg/ml bovine serum albumin, 50% glycerol. Store
in small aliquots at -20°C.
- CsCl solutions for banding: Transfer the indicated
amounts of analytical grade CsCl into small beakers to
give the desired final densities:
final solution (g/ml)
of CsCl (g)
Add 100 ml 10 mM Tris-HCl, pH 8, to each beaker and
stir to dissolve. Verify density by weighing 1.0 ml of
each solution (e.g., 1.0ml of the 1.35d solution
should weigh 1.35 g). Sterile filter and store at room
- Sterile glycerol: Prepare by autoclaving.
- TE/SDS: Add 0.5ml 20% SDS to 100ml 10mM Tris-HCl, 1 mM EDTA, pH 8.
- Grow 293N3S cells in spinner culture to a density
of 2-4 × 105 cells/ml in 3 liters of complete Joklik's
modified MEM + 10% HS. Centrifuge cell suspension
at 750g for 20min, saving half of the conditioned
medium. Resuspend the cell pellet in 0.1 vol fresh
medium and transfer to a sterile 500-ml bottle containing
a sterile stir bar.
- Add virus at an MOI of 10-20PFU/cell and stir
gently at 37°C. After 1 h, return culture to the spinner
flask and bring to the original volume using 50% conditioned
medium and 50% fresh medium. Continue
stirring at 37°C.
- Monitor infection daily by inclusion body staining
- Remove a 5-ml aliquot from the infected spinner
culture. Spin for 10min at 750g and resuspend
the cell pellet in 0.5 ml of 1% sodium citrate.
- Incubate at room temperature for 10 min, add 0.5
ml Carnoy's fixative, and fix for 10min at room
- Add 2ml Carnoy's fixative, spin for 10min at 750 g, aspirate, and resuspend the pellet in a few
drops of Carnoy's fixative. Add one drop of fixed
cells to a slide, let air dry for about 10min, add
one drop orcein solution and a coverslip, and
examine in the microscope. Inclusion bodies
appear as densely staining nuclear structures
resulting from the accumulation of large
amounts of virus and viral products at late times
in infection. A negative control should be
included in initial tests.
- When inclusion bodies are visible in 80-90% of
the cells (1.5 to 3 days), harvest by centrifugation at
750g for 20min in sterile l-liter bottles. Combine
pellets in a small volume of medium and spin again.
Resuspend pellet in 15 ml 0.1M Tris-HCl, pH 8.0. Store
at -70°C until use.
- Thaw the frozen crude stock and add 1.5 ml 5%
Na deoxycholate. Mix well and incubate at room temperature
for 30min. This disrupts cells without disrupting
virions, resulting in a relatively clear, highly
- Add 0.15ml 2M MgCl2 and 0.075ml DNase I
solution and then mix well. Incubate at 37°C for 60min, mixing every 10min. The viscosity should be
- Spin at 3000g for 15min at 4°C in a tabletop centrifuge.
- Prepare three CsCl step gradients in SW41 Ti
ultraclear tubes: Add 0.5ml of 1.5d CsCl solution to
the bottom of each tube, carefully overlay with 3.0ml
of the 1.35 d solution, and then overlay with 3.0ml of
the 1.25d solution. Mark the level of the interface
between the 1.35d and the 1.25 d layers.
- Carefully add 5 ml of supernatant from step 6 to
the top off each gradient. If necessary, top off tubes
with 0.1M Tris-HCl, pH 8.0.
- Spin at 35,000rpm in a Beckman SW41 Ti rotor
at 10°C for 1 h.
- Collect the virus band at the interface between
the 1.35d and the 1.25d layers by piercing the side of
the tube with an 18-gauge needle attached to a 5-ml
syringe. Pool the virus from all three tubes into a
Beckman SW50.1 ultraclear tube. If necessary, top off
the tube with 1.35d CsCl solution.
- Centrifuge the pooled virus in a Beckman
SW50.1 rotor at 35,000rpm, 4°C for 16-20h.
- Collect the virus band in the smallest volume
possible, transfer to a Slide-A-Lyzer dialysis cassette,
and dialyze at 4°C against three changes of 500
volumes 10 mM Tris-HCl, pH 8.0, for at least 24 h total.
- After dialysis, add sterile glycerol to a final concentration
of 10%. Store the purified virus at -70°C in
- Determine titer of virus by plaque assay
- The concentration of virus particles, based on
DNA content, can be determined spectrophotometrically
- Dilute purified virus 20-fold with TE/SDS. Set
up blank by diluting virus storage buffer (10
mM Tris, pH 8.0, supplemented with glycerol to
10%) 20-fold with TE/SDS.
- Incubate for 10min at 56°C. Vortex sample
briefly. Measure OD260 with a spectrophotometer.
- Calculate the number of particles per milliliter,
based on the extinction coefficient of wild-type
Ad as determined by Maizel et al. (1968) as
particles/ml = (OD260)(20)(1.1 × 1012)
Once the desired recombinant Ad virus is obtained,
the ability to express the foreign gene must be tested. The most suitable procedure for detecting expression
would depend on the particular properties of the
foreign protein. If antibodies to the protein are available,
then ELISA, Western blotting analysis, or
immunoprecipitation of infected cell extracts may be
the simplest method to quantitate protein expression.
If possible, the biological activity of the recombinant
protein should also be tested to ensure that the
expressed protein is functional (for additional details,
see Graham and Prevec, 1991).
It is important to use caution when handling recombinant
Ads. Experimentation with these vectors
should be carried out in accordance with relevant regulations.
If exposed inadvertantly, individuals without
previous immunity to Ad5 may seroconvert not only
against Ad5, but also against the foreign gene product
expressed. This should be avoided, especially if the
development of antibody may confuse diagnosis of a
particular disease. Finally, no toxic or oncogenic gene
product should be expressed from replicationproficient
Ad vectors without an appropriate increase
- There can be a number of different causes for
failure to obtain the proper recombinant virus following
cotransfection. First, the transfection efficiency may
be low (a suitable control would be transfection of
wild-type viral DNA or infectious plasmid DNA such
as pFG140). The 293 cells used in transfections must be
at low passage, growing slowly, and slightly subconfluent.
In addition, the plasmid DNA must be of high
quality; we routinely use CsCl-banded DNA in
cotransfections. Finally, although infrequently, the
desired recombinant might not be obtained because the
foreign gene insert is toxic to the cells or virus, in which
case it may be necessary to use an alternate adenovirus
rescue system (e.g., Hi-IQ AdMax from Microbix).
- For many applications, particularly those involving in vivo studies or infection of human cells, it is
important to confirm that preparations of E1 replacement
Ad vectors are not contaminated with replication-
competent Ad (RCA). Most often RCA are
generated by recombination of the vector with E1
sequences in the 293 cell genome. The latter is most
problematic when the wild-type Ad has a growth
advantage over the desired recombinant virus. Several
tests for RCA contamination have been described,
including a functional assay for virus growth in noncomplementing
cell lines such as HeLa or A549, as well
as protocols that detect contaminating wild-type viral DNA sequences, such as Southern blot hybridization
analysis and quantitative polymerase chain reaction
amplification (Lochmuller et al., 1994). It is advisable
to perform at least one of these tests in order to estimate
the maximum possible RCA contamination in
recombinant virus stocks before use.
- The level of expression in E1 replacement vectors
depends mainly on the strength of the promoter immediately
upstream of the coding sequence for the
foreign gene. In E3 insertion vectors, this is not necessarily
true; even some promoterless constructs express
relatively high levels of recombinant proteins. In these
cases the major late or E3 promoter is presumably
driving expression of the foreign gene insert. It is not,
at this time, possible to predict which E3 constructs
will utilize an inserted promoter; consequently,
care must be taken in analyzing E3 insertion recombinants
for appropriate expression, in particular, for
example, when transcriptionally regulated expression
We thank Urea Sankar, John Rudy, and Derek Cummings
for excellent technical assistance. This work was
supported by grants from the National Institutes of
Health, the Canadian Breast Cancer Research Initiative,
the Canadian Institutes of Health Research
(CIHR), and the National Cancer Institute of Canada.
P.N. was supported by a CIHR Postdoctoral Fellowship.
Addison, C. U, Hitt, M., Kunsken, D., and Graham, E L. (1997).
Comparison of the human versus murine cytomegalovirus
immediate early gene promoters for transgene expression by
adenoviral vectors. J. Gen. Virol
Berkner, K. L. (1988). Development of adenovirus vectors for expression
of heterologous genes. Biotechniques 6
Bett, A. J., Haddara, W., Prevec, L., and Graham, E L. (1994). An efficient
and flexible system for construction of adenovirus vectors
with insertions or deletions in early regions 1 and 3. Proc. Natl.
Acad. Sci. USA 91
Bramson, J., Hitt, M., Gallichan, W. S., Rosenthal, K. L., Gauldie, J.,
and Graham, E L. (1996). Construction of a double recombinant
adenovirus vector expressing a heterodimeric cytokine: In vitro
and in vivo
production of biologically active interleukin-12. Hum.
Ginsberg, H. S. (1984). "The Adenoviruses." Plenum, New York.
Graham, E L., and Prevec, L. (1991). Manipulation of adenovirus
vectors. In "Methods in Molecular Biology"
(E. J. Murray, ed.), Vol.
7, pp. 109-128. Humana Press, Clifton, NJ.
Graham, E L., and Prevec, L. (1992). Adenovirus-based expression
vectors and recombinant vaccines. In "Vaccines: New Approaches
to Immunological Problems"
(R. W. Ellis, ed.), pp. 363-389. Butterworth-
Heinemann, Boston, MA.
Graham, F. U, Smiley, J., Russell, W. C., and Nairn, R. (1977). Characteristics
of a human cell line transformed by DNA from human
adenovirus type 5. J. Gen. Virol. 36
Hitt, M. M., Parks, R. J., and Graham, E L. (1999). Structure and
genetic organization of adenovirus vectors. In "'The Development
of Human Gene Therapy"
(T. Friedmann, ed.), pp. 61-86. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Lochmuller, H., Jani, A., Huard, J., Prescott, S., Simoneau, P. M.,
Massie, B., Karpati, E, and Acsadi, G. (1994). Emergence of early
region 1-containing replication-competent adenovirus in stocks
of replication-defective adenovirus recombinants (AE1 + AE3)
during multiple passages in 293 cells. Hum. Gene Ther
Maizel, J. V., White, D., and Scharff. M. D. (1968). The polypeptides
of adenovirus. I. Evidence of multiple protein components in the
virion and a comparison of types 2, 7a, and 12. Virology 36
Ng, P., Cummings, D. T., Evelegh, C. M., and Graham, E L. (2000b).
Yeast recombinase FLP functions effectively in human cells for
construction of adenovirus vectors. Biotechniques 29
Ng, P., and Graham, E L. (2002). Construction of first-generation
adenoviral vectors. Methods Mol. Med
Ng, P., Parks, R. J., Cummings, D. T., Evelegh, C. M., and Graham,
E L. (2000a). An enhanced system for construction of adenoviral
vectors by the two plasmid rescue method. Hum. Gene Ther
Sambrook, J., and Russell, D. (2001). "Molecular Cloning: A Laboratory
Manual" 3rd Ed. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY.