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3. I. 1. Choice of Viral Background
The degree of attenuation of the vnus needs to be considered in choosmg a
parental strain or viral background. If the vector is intended for human use, an
extremely attenuated virus will be required, which will probably involve mak-
mg the virus completely unable to replicate so that a helper cell lure 1s required
for propagation. Some deletions that give rise to a nonreplicatmg virus are
ICP4 (major viral transacttvator, which is required for immediate early and
early gene functtons) or DNA polymerase. The protocol below uses an ICP4
deletion vn-us as an example. This mutant has the advantage over the poly-
merase deletion of being very restricted in overall expression of other viral
genes, thus minimizing cytopathic effects. If the virus is not intended for human
use and is going to be used m animal models, more choices are avatlable.
Consideration must be given to whether the vn-us will be maculated into the CNS
373
HS V Vectors
or periphery. An advantage of HSV vectors is that a great deal is known about
pathogenic parameters. If the virus is to be used in the periphery, HSVl stram
KOS (see Note 16) can be used without f%rther attenuation, smce this virus is not
neuroinvasive and will not spread through the nervous system (17). However, if
one 1sInterested in the CNS, then thusstrain 1svirulent and will kill tf inoculated
directly into the brain. HSV can be easily attenuated with respect to virulence by
either insertion of the desired gene into the tk gene (18), ribonucleotide reductase
(19), or dUTPase gene (20). Inactivation of these genes allow the virus to reph-
cate to near-normal levels in rapidly dividing cells (including cell culture), but
not within the nonreplicating neurons of the nervous system. In addition, use of
the tk gene allows one to select for recombmants based on the tkphenotype if a tk
cell line (such as rat-2) is used. Another example is the nonessential glycopro-
tein, gC (26,22). Figure 1 (p. 374) illustrates the location of a number of the
commonly used msertion sites m the HSV genome; Table 1 (p. 375) compares
the pathogenic properties of virus with mutations in these genes. The process of
transfecting and screening for viral recombinants is ilhstrated m Fig. 2 (p. 376).
3.1.2. Design of the Recombination Plasmid
Once one has determined which gene will be used as the insertion site, it is
relatively easy to destgn a recombination plasmid. If the desired gene 1sto be m-
set-ted the tk or ICP4 gene, anumber of insertion plasmids are available, or one
into
can be easily generated. This is facilitated by the fact that the entire nucleotide
sequenceof HSV (stain 17+) is known (27). The only important pomt to keep in
mind is that one needsat least 200 bp on either side of the insert to obtain homolo-
gous recombmation of the gene onto the vu-al genome. This ISvery mmtmal, and 1
kb on either side is recommended for optimal recombination efficiencies
3.1.3. Engineering the Promoter
The promoter that is used here will depend on the duration of expression
that is desired. If in vitro expression for only several days is required, then an
ICP& vector using almost any cellular promoter will be sufficient. The same
holds true for short-term expression (Cl4 d) within animals. However, if long-
term expression of genes m the animal is required, then the binary LAT-LTR
promoter is recommended. A schematic of an ICP4 recombination plasmid
utlizmg this promoter is illustrated in Fig. 3 (p. 377).
3.2. Consfrucflon of the Viral Recombinant
Once the recombmation plasmid has been constructed, the recombinant virus
can be constructed. This is performed by cotransfectmg HSV-DNA (whrch IS
infectious) with the recombination plasmid DNA.
374 Bloom
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I 0
E
2
HSV Vectors 375
Table 1
Comparison of the Virulence Properties
of HSV-1 Mutants Commonly Used as Vectors
Essential gene,
LDs,,/PFU helper cell
Viral insertion sate Peripheral line required Refs.
IC

7.22
ICP4 (IE 175) transcriptional >10* >10* Yes
activator
Thymidme kmase (UL23) 1 x 10s >10* No 23
Ribonucleotide reductase 105 n.d. No 8,19,24,25
(UL39)
20
dUTPase (UL50) 3 x 102 3.5 x 106 No
No 8
Protein kmase (US3) 104 n.d.
Glycoprotein C (UL44) >10*
103 No 12,21
17+ (wild-type) 4 104 N/A 26
KOS (wild-type) 25 >10* N/A 26


3.2.1. Preparation of HSV Transfection DNA
1 Trypsmize five confluent T75 flasks of rabbit skm cells, and resuspend each flask
in a total of 15 mL of MEM. Seed lo- to 150-mm dashes with 7 mL of this cell
suspension by adding the cells to 20 mL of supplemented media m each dish
Incubate overnight at 37°C.
2. On the followmg day (the dishes should be approx 90% confluent at this point)
the media are removed, and the cells are infected by adding 5 mL of media con-
taining 2 x lo6 PFU of HSV The vnus is allowed to adsorb to the cells for 60 min
at 37°C The dishes are rocked gently halfway through the incubation.
3 After 1 h, 25 mL media are added to the cells, and the dishes Incubated until all of the
cells have rounded and detach easily when the dish is swirled This usually takes 2-3 d
4. Harvest the cells by pipeting the cells off the bottom of the dishes. Transfer the
cell suspenston to Sorvall bottles, and centrifuge at 10,000 rpm at 4°C for 40 mm
5. Pour off supernatant, and resuspend the pellet in hypotonic lysis buffer (10 mL)
and transfer to a comcal 15-mL falcon tube. Vortex vigorously and incubate for 5
min on ice. Vortex again briefly
6 Centrifuge at 8OOg for 10 mm at 4°C (this pellets the nuclei)
7. Transfer the supernatant to a new conical tube, and add 1 mL 10% SDS and 0 5
mL 20 mg/mL pronase
8 Incubate for 1 h at 50°C
9. After 1 h, add another 0 5 mL of 20 mg/mL pronase, and mcubate overnight at 37°C
10. Phenol extract 2X
11. Phenol/SEVAG (1 1) extract 1X.
12. SEVAG extract 1X.
13. Dialyze vs 1X TE overnight at 4°C (with 2 changes of buffer).
376 Bloom
8
F
t
2
HSV Vectors 377




Fig. 3.ZCP4 insertion vector.


14. Determine the concentration of DNA spectrophotometrically by determining the
absorbance (AZ&.
15. Digest 1 pg of DNA with HindHI, and run on an agarose gel along with uncut
DNA to determine the purity and quality of the preparation. There will be some
cellular contamination, but the viral DNA should be the predominant form, and
there should be little evidence of smearing.
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16. For long-term storage of the DNA, it IS advisable to ahquot the DNA into small
fractions and freeze Repeated freeze-thawing should be avoided
3.22. Transfect˜on of HS V-DNA
Transfectrons are performed m 60-mm dishes on subconfluent monolayers
of rabbit-skin (RS) cells. The RS cells are propagated m MEM supplemented
with 5% calf serum and glutamme, Penn/Strep Unit-length HSV-DNA IS
cotransfected with the desired plasmid at various ratios usmg a modified cal-
cmm phosphate precipitation procedure The transfections are generally
allowed to proceed until all all of the cells are rounded, i.e., 100% cytopathrc
effect (CPE) 1s evident (usually 34 d), though the dishes may be harvested
earlier rf one wishes to prevent ampltfication of siblings.
1. 60-mm Dishes are seeded from a flask of actively growing RS cells at a ratio that
will produce a cell density of approx 50% confluence the followmg day (typically,
1/30of a T75 flasW60mm dish) The dishes are Incubated O/N at 37°C 5% CO,
2 On the followmg day, the media are removed from the dishes (which should be at
50% confluence) and replaced with MEM supplemented with 1 5% fetal bovine
serum (FBS) The dishes are then Incubated O/N at 31 5°C 5% CO, This IS to
serum-starve the cells
3 The transfectron mix is prepared by dilutmg the desired amount DNA (typically,
l-10 pg of HSV/dish, and a lo-fold molar excess of the lmearrzed plasmid DNA)
m a final volume of 225 pL of TNE buffer After the dilutions have been made,
25 PL of a 2 5MCaC1, is added to each tube (see Notes 1 and 2)
4. The DNA is precrpitated by adding 250 PL of 2X HEPES buffer to the above
sample while bubbling an mto the solution with a mouth aspirator connected to a
sterile-plugged Pasteur prpet
5 The solution is then Incubated for 20 mm at room temperature
6. Aspirate the 1 5% FBS MEM from the 60-mm dishes, and pour on the transfec-
tion mix. Incubate the dishes at room temperature for 20-30 mm
7 Add 5 mL of 1 5% FBS MEM, and incubate for 4 h at 37°C. Do not remove the
DNA solution.
8. After 4 h, aspirate the media, and wash the monolayer with media two times
Then hypertonic shock the cells briefly (˜1 mm) by addmg l-2 mL of shock
buffer (1X HEPES, 20% dextrose solution).
9. Aspirate the shock buffer, and wash 2X with media After the last wash, add
5 mL of MEM 5% calf serum to the dishes, and incubate 34 d at 37°C 5% CO,
10 The transfections are harvested by scraping into the media with a rubber
policeman Recombmants are screened by plaqulng the cells on RS cells,
and picking the plaques mto 96-well dish to which media have been added
to the wells These dishes are frozen, and 50 pL of each well used to infect
96-well dishes of confluent RS cells (see Note 5). These dishes are then
dot-blotted and probed with the desired insert Typical transfections yield
2-10 positives/96-well dish.
379
HS V Vectors
3.2.3. Plaquing of Transfections for Recombinants
1. Transfection mixes are plated onto confluent monolayers of rabbtt skm cells
m 60- or loo-mm dishes Generally, from a transfectton that was performed
in a 60-mm dish that was allowed to go to 100% CPE, dilutions of 1O-5 or
10m6yield well-tsolated plaques that are suttable for prcking The infected
monolayers are overlayed wtth 0.4% (final) agarose in 1X supplemented
medta, and incubated for 2 d
2. On the mornmg of the third day, the dishes are counterstamed with neutral
red to atd m the vtsuallzatton of the plaques A l-30 dilution of the Neutral
red-stock solution IS made in unsupplemented media. An equal volume of
the neutral red overlay 1sthen added to the dashes on top of the agar overlay
(for 60-mm dishes, 5 mL of diluted neutral red are added to each dish), and
the dishes are incubated at 37™C until the monolayers are stamed red For
RS cells, this IS approx 6 h.
3. After the monolayers are stained, the liquid overlay is aspirated and the plaques
are picked usmg a sterile Pasteur pipet. The plaques are picked by applymg slight
pressure to the bulb of the plpet, then coring the plaque straight down, and twlst-
mg the ptpet The bulb 1sthen released, and the plaque aspirated partially mto the
pipet. The plaque IS then expelled into a well of a 96-well dish that has been filled
with two to three drops of media.
4. After all of the plaques are picked, the dish is frozen at -70°C and then thawed m
the Incubator.
5. The plaques are then amplified by plating onto a 96-well dish of confluent RS
cells. The media 1s“flicked” off the dish, and using a multichannel pipeter, 50 pL
of the wells with the plaques are transferred to the 96-well plate with the RS
cells. The vnus is then allowed to adsorb for 1 h at 37°C. At the end of the ad-
sorption period, two drops of supplemented media are added to each well, and
the dishes incubated until the wells show 100% CPE (usually 3 d)
3.2.4. Screening for Recombinants
1 After cells in the wells of the 96-well dishes have reached full CPE (usually 3-4 d),
they are ready to be dot-blotted.
2 Set up the M&pore dot-blot apparatus with one piece of blottmg paper underneath a
piece of nylon membrane (Hybond-NTM or NytransTM) Wet the blotting paper and
membrane completely with 2X SSPE before clamping the apparatus together
3. After clamping the apparatus together, apply vacuum Using a multichannel ptpeter,
transfer 50 PL of the infected cells from each well of the 96-well dish to the appa-
ratus (pipet the wells up and down several times to mix before transferring)
4. After the media have filtered through the apparatus, add 200 pL of solution A to
each well of the apparatus
5 Likewise, after solution A has filtered through all of the wells, add 200 pL of
solution B.
6. Finally, after all of solutron B has filtered through the apparatus, add 200 pL
of solutron C.
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7. Remove filter from apparatus, label the filter (remember to mark orientation),
and bake at 80°C for 1 h The blot IS now ready for hybridization (see Note 3).
8 Freeze the 96-well dish at -70°C for later use.
3.2.5. Confirmation of Viral Recombinants
After the vu-us stocks have been plaqued-purified for at least three rounds, a
small stock of several “clones” can be grown up for confirmation. Typically,
the followmg tests are performed using the standard techniques of viral genome
and RNA analysts presented elsewhere in this volume.
1. Southern blot analysis: The goal IS to determme that the gene of interest IS inserted
into the proper location within the viral genome If the recombination site is
within the viral repeat sequences, it 1s important to deterrnme that the vu-us 1s
“double-srded” or that two copies of the gene are present This is critical in that
single-sided vu-uses tend to be unstable, and the inserted gene may recombine
out. In addition, if the construct has been inserted into the ICP4 gene, both copies
must be deleted in order to attenuate the virus
2 Northern blot analysis. This is performed in order to determine that the transgene
IS transcribed properly in the context of the viral genome.

3.3. In Viva Testing of Recombinant Virus
If the goal is to utilize the recombinant vu-us to express a transgene m
vivo, there 1s no reliable m vitro method for determinmg whether a par-
ticular construct will express the desired gene durmg latency and deter-
mining levels of expression in the desired cells, so it is necessary to test
recombmants m VIVO. Presented below is the methodology for evaluating
the ability of an HSV recombinant to express a gene within the spmal
ganglia of mice.
3.3.1 Testing for Expression in Mouse Sensory Ganglia
For studies involvmg expression within the penpheral nervous system, estab-
hshment of a latent infection within the mouse lumbosacral ganglia provides an
efficient model for assessing the expression and/or evaluating the biological effects
of a given recombinant. Mice can be easily infected by footpad inoculatton. Even
nonrephcating vectors can be evaluated m this manner, because a productive infec-
tton 1s not required for the establishment of a latent infection in these ganglia. In
fact, all that is needed is to expose the virus to peripheral nerve termiru, which can
be performed by lightly abrading the rear footpads. In general, a period of 2-3 wk
IS required prior to assessmg latent expression, since this is the amount of time that
it takes for all traces of acute infection to subside (see Notes 8 and 9). After this
penod of time, the mice can be sacrificed, and the ganglia removed and subjected
to a number of different histologic or biochemtcal analyses.
HS V Vectors 381

3.3.1 .l . FOOTPAD INOCULATION OF MICE
3.3.1.1.1 Saline Pretreatment
1 Six-week-old outbred Swiss-Webster mice (16-19 g) are anesthetized one at a
time briefly in an ether (or Halothane) Jar (Just until heavy breathing ensues)
(See Note IO.)
2. The mice are injected with a sterile 10% saline solution under the footpad of each
foot with a l-cc tuberculin syringe with a 28-gage needle Approximately 0.1 cc/
foot IS injected (until it IS swollen).
3. The mice are returned to their cages and left for at least 4 h (but no longer than 6).
4. The mice are now ready for infection.
3.3.1 1.2. Inoculation

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