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origin of replication and the ampicillin resistance gene.
2 Sequences from HSV that support propagation of the amplicon m vnus stocks
These include an HSV origin of replication (ori)-orzS or orlL (25,28), and the
two HSV packaging signals, pat-1 and pat-2, located within the “a” sequence of
the HSV-1 genome. The pat-1 and pat-2 are consensus sequences of all her-
pes-viruses, which define distance and structure of the corresponding genomic
termini (Delss and Frenkel, 1986; Deiss et al, 1986).
3. A transcriptlon unit that includes a promoter element (e.g., HSV-1 munedlate
early [IE] 4/5 promoter [2/ and CMV promoter [15/) This is then followed by an
expressed foreign gene and the SV40 early region polyadenylation site
Consideration of c&acting functions required for ampllcon DNA propa-
gation, includes the HSV origin (ori) of DNA replication that can be van-
able. HSV contains two types of origins of replication: the or& two copies
of that are located within the c reiterated sequence of the S component, and
the oril-one copy of which is mapped in the middle of the L component.
By comparison, both S and L ortgins contain ori binding proteins-two cop-
ies of that are present in both the S ori and the L ori. The oriL IS slightly
longer because of A + T-rich perfect palmdrome sequences, which make it
unstable m DNA fragments cloned in E. colz. Both types of origins are suffi-
cient for propagation of standard HSV inasmuch as both copies of oriS have
recently been deleted without impairing the ability of the vu-us to multiply
(29). In terms of oriS and oriL, they were both shown to provide proper ori-
gins for amplicon replication (1,29,30).
The promoter element, HSV IE promoters in the amplicon-based vectors are
not subjected to the promoter inactivation that occurs with many types of virus
vectors, including defective HSV vectors (31). HSV IE promoters can support
expression in human, monkey, and mouse in vitro, which is very important,
because it is needed for preparations of stocks to be used m different animals m
vivo and in gene therapy (27,32). The cytomegalovirus (CMV) IE promoter
can also directly express foreign genes, but downregulation of the expressed
gene after 2 wk was reported in in vivo experiments (15,Z7). Promoters from
housekeeping genes may be more effective at causing sustained changes in
central nervous system function. Cell-type-specific promoters may allow
restricted expression of a gene in the vector to a chosen cell type.
230 Frenkel and Sand

The gene m the amphcon can be subjected to experimental manipulation.
The E coli 1acZ gene could be expressed and serve as a marker gene employ-
mg in sztu /3-galactosidaseactivity assay.This gene can be replaced with virtu-
ally any gene. In addmon, by fusing the gene to subcellular targeting sequences,
the expressed protein can be localized to a particular part of the cell One can
construct a plasmid that contams the IacZ gene as a marker gene together with
a cellular gene under different promoters.
There is a limit to repeat unit sizes. We have found that constructed
amplicons with repeat units below 15 kbp were stably propagated, whereas
amphcons with larger seed repeats underwent systematically random deletions,
generating deleted repeats smaller than 15 kbp, which thereafter retained their
stability (32). The reasons for this limitatton are as yet unclear, but they may
have to do with rollmg circle rephcatton.
1.2. Helper Virus Properties
In terms of the helper virus, the orrgmal work with the amplicon has been
done with nondefective helper virus, as well as a temperature-sensitive (ts)
mutant defective with respect to ICP4 (2,26). The wild-type HSV-1 m the virus
stock mvarrably causes cell death. However, mtracerebral mjection (33) and
infection of mouse neuroblastoma cells with HSV-1 ts mutants allow persis-
tence of the vu-us without cell death (23). The ts mutants are affected m the
ICP4 gene (also termed IE3,Vmw 174), the IE gene responsible for turning on
P-gene expression, and hence a critical turning point in the cascade regulation
of HSV gene expression m the infected cells (34). At the restrictive tempera-
ture of 37 or 39°C the ts mutations block the lytic cycle and thereby prevent
cell damage. Virus is grown at the permtsstve temprature of 3l-34°C.
Deletion mutants are better than ts mutants as the helper, resulting m lower
frequencies of reversion to wild-type (26,27). An ICP4 gene deletion mutant,
D30EBA (35), which requires for its propagation an ICP4-containing cell line,
has a reversion frequency of 5 x 10M5 compared to the reported reversion fre-
quency of t.sK- 2 x 10e3.By using HSV-1 deletion mutant viruses (grown on
complementing cells), it is possible to obtain vu-us stocks that are unable to
replicate on normal noncomplementing cells. Additional helper viruses are
currently beemg developed (36).
1.3. Amp/icon Propagation: Preparation and Analyses
of Transfection-Derived Virus Stocks
DNA transfections are done using rabbit skin (RS) cells, whereas the virus
is grown in Vero (African Green Monkey kidney) or Hep-2 (Epidermoid carci-
noma) cells. Mixtures of plasmid DNA and HSV- 1 helper virus DNA are used
to transfect RS cells in 25-cm2 flasks by the calcium-phosphate coprecipitation
HS V Amp/icons 231

technique (3 7,381.Transfectron efficiencies should be first determined by estt-
mating the proportron of blue cells/total cells after transfectmg with plasmtd
containg P-galactosidase driven by an adequate promoter. Alternatively,
amplicon DNA can be transfected followed by supermfectron with the helper
vn-us. The derived virus stocks are harvested by three cycles of freeze (-SO™C)
thawing (37°C). The virus stocks derived from the cotransfectton or transfec-
tron-superinfection (passage 0) are serially passaged, taking a fixed ratio for
each passage (e.g., I :4 dilution) in Vero or Hep-2 cells, to generate subsequent
virus stock passages. Propagation continues while determinmg the ratio of
defective:helper virus in each passage. Required passage numbers depend on
the amplicon and helper vnus employed. The choice of passage to be used as a
constructed amplicon stock depends on the amount of foreign gene expression
aimed for. Concentrations of the helper virus are determined by titration plaque
assay.Defective vnus can be assayedm culture via htstochemical detection of
bacterial P-galactosidas-ach blue cell 1sconsidered to represent one infec-
tious defective virron.
2. Materials
2.7. DNA Transfection of RS Cells
1 RS cells (Epidermis, cottontail rabbit): cells are available from the American
Type Culture Collection as CCL-68.
2 Hep-2 cells (Eptdermoid carcmoma)™ cells are available from the American Type
Culture Collection as CCL-23.
3. Vero cells (African Green Monkey kidney): cells are available from the Ameri-
can Type Culture Collection as CCL-8 1.
4 DMEM: Dulbecco™s modified munmum essential medium.
5. Maintenance medium: DMEM supplemented with 10% inactivated fetal calf serum
(IFCS-30 min at 56°C).
6. 199-V medium-Ml99 (Hank™s) medium containing 1% inactivated calf serum
(ICS-30 min at 56°C) and 100 U/mL penicillin/100 mg/mL streptomycm.
7 Sterile microtubes
8 2X HeBSa Dissolve 8 2 g of NaCl, 5.95 g HEPES, and 0.105 g Na2HP04 m
double-distilled (dd) Hz0 to a final volume of 500 mL. Bring to pH 7.05 with 5N
NaOH. Sterilize by filtering through 22-urn filter, and store at -20°C.
9. 2M CaC12* sterilize through 22-urn filter, and store at -2O“C.
10 Sterile ddHzO.
11 Sterile glycerol.
12 Amplicon and helper vnus DNAs/helper virus stock

2.2. X-Gal-Based Histochemical Reaction
1. Stocksolutronsof lMNa2HP04, 1MNaH2P04,and lMMgC&* Preparein ddH,O
and store at room temperature.
Frenkel and Sand
232

2 Stock solutions of 50 tipotassmm ferrlcyamde (K,Fe[CN],) and 50 mMpotas-
slum ferrocyamde (K,Fe[CN],)* Prepare m ddH,O and store m foil-wrapped
glassware (in the dark) at 4”C, where they are stable for at least 3 mo
3. X-gal stock: Dissolve in NJ-dimethyl formamide at 20 mg/mL, and store in a
glass container (not polycarbonate or polystyrene) m the dark at -20°C.
4 4% Paraformaldehyde (wear a mask and gloves when handling paraformalde-
hyde). In a fume hood, dissolve 8 g of powder m 150 mL 0 1M sodium phos-
phate, pH 7.3 (80 mMNa,HPO,, 20 mMNaH,PO,) while stirring and heating to
60°C. Add 1ON NaOH at a rate of 1 drop/min until the solution clears Bring the
volume to 200 mL with 0 1M sodium phosphate, pH 7 3 Store at 4°C for up to 1 mo
5 25% Gluteraldehyde.
6 Working fixative (2% paraformaldehyde, 0.2% gluteraldehyde m O.lM sodium
phosphate): combme 50 mL of 4% paraformaldehyde with 49.2 mL of O.lM
sodium phosphate, pH 7 3, and 0.8 mL of 25% gluteraldehyde This can be stored
at 4°C for up to 1 wk.
7. X-gal stain: 100 mA4 sodium phosphate, pH 7.3 (80 mM Na,HPO,, 20 mM
NaH,PO,), 1 3 rnA4MgCl,, 3 mA4K,Fe[CN16, 3 mMK,Fe[CN],, and 1 mg/mL
X-gal Filter through a 0 45-p disposable filtration umt prior to use
8 Phosphate-buffered salme (PBS)* 15 mMsodmm phosphate, pH 7 3,150 mA4NaCl

3. Methods
3.1. DNA Transfection of RS Cells
1 Prepare RS cell cultures in 25-cm2 flasks
2 Decant the medium and overlay with fresh mamtenance medium.
3. Preparation of DNA/CaCl reaction: For each transfectlon prepare two sterile
microtubes In the first tube, put 250 pL of 2X HeBS; m the second tube, which 1s
kept cold m ice, put 30 mL of 2M CaC12, DNA, and ddH20 to a final volume of
250 pL Add the DNA mixture to the 2X HeBS, mix gently (do not vortex), and
allow to precipitate at room temperature for 20 mm
4. Add transfectlon mixture onto the RS cells monolayer, apply to the middle of the
monolayer and ensure that it is properly distributed
5. Incubate at 37°C for 4-6 h.
6 Remove the medium, and add 2 mL DMEM containing 10% glycerol Incubate
for 1 min, and quickly remove the medium. Rinse twice with DMEM The glyc-
erol-containing medium is toxic to the cells and should not be left m contact with
the cells for longer times
7. Overlay with maintenance medium. Incubate for 4-5 d, or until the viral mfec-
tlon 1sspread rn the entire culture
3.2. X-Gal-Based Histochemical Reaction-Detection
of Defective Viruses with IacZ Bacterial Gene as a Marker
1. Remove media
2. Rinse the cell monolayer twice with PBS.
3. Overlay the culture with the fixative. Incubate at 4°C for 5 min.
233
HS V Amp/icons
4. Remove the fixative, and rinse twice with PBS at room temperature.
5. Remove PBS and overlay the fixed cells with the X-gal stain. Incubate at 37™C
until blue color develops (30 min to overnight).

4. Notes
1. The amplicon plasmid can contain either the or& and oriL, which are both func-
tional. The oriL sequences are less stable in DNA fragments cloned in E coli
2. Amplicon propagation could begin with cotransfection of the cells with
amplicon and helper vu-us DNA or, alternatively, tt could involve transfection
of the cells with amplicon DNA followed by superinfectton with the helper
virus. For transfectton-superinfectton, begin by transfecting the cells with
amplicon DNA, and follow by infection with the helper virus at a MO1 of 1
PFU/cell. Incubate in 199-V medium.
3 The amphcon virus stocks containing defined ratios of helper virus and amphcon
can be repeatedly reproduced.
4. It is most important to change pipet or tips for each dilution to prevent carryover
of virus, which wtll result in anomalous high titers. Make a short vortex before
using the virus and after each dilution Pipet up and down because the vu-uses
adhere to the plastic.
5. The titers of the defective vu-us stocks can be assayed in culture via histochemi-
cal detection of bacterial P-galactostdase Alternatively, serially passaged viral
DNA can be 32P-labeled, and restriction enzyme analyzes can be done by
eletrophoresis m agarose gel (1)

Acknowledgments
We thank The Isreal Academy of Science, The Isreal Health Ministry, and
The Wolfson Family Charitable Trust.
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