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Fig. 5. The specificity of the in situ PCR was confirmed by Southern hybridization.
DNA was extracted from a single 6-p section aftergB in situ PCR, and hybridized with
a [y32P]-labeled gB oligonucleotide probe that recognized a region of the LAT product
between the pair of primers used for amplification. Lane 1, DNA from an 8-wk
infected ganglionic section after amplification; lane 2, DNA from an uninfected gan-
glionic section after amplification; lane 3, control 191-bp gB fragment.

d. Find the optima1 amount of target DNA.
e. Use the minimum number of cycles possible. We use not more than 30.
2. Competitive quantitative PCR:
a. Ensure that the restriction enzyme digestion is complete. Be sure to run controls.
b. Dilute the product of the first coamplitication at least 200: 1 to avoid hetero-
duplex formation.
3. Competitive quantitative RT-PCR:
a. The most important point is to ensure that the DNase treatment is complete.
Be sure to run controls in which reverse transcriptase is absent.
In situ PCR:
4.
a. In our experience, perfused paraffin sections work the best.
b. Vary the number of cycles to find the appropriate number that gives the best
signal-to-noise ratio.
c. Observe the same precautions as noted above for PCR.
5. Normalization of DNA and RNA extracts:
Ten microliters of each extract can be amplified using primer pairs for the
cellular glyceraldehyde phosphate dehydrogenase (GMDZf,, gene in the pres-
ence of [a32P]dCTP as tracer, to ensure that the extracts contain equivalent
amounts of DNA, using the same conditions described for gB amplification. If
necessary, the amounts of DNA can be adjusted.
In order to normalize the amount of starting RNA, lo-pL aliquots of each
RNA extract can be amplified by RT-PCR using primer pairs for the GAPDH
transcript, using the same conditions described for LAT RT-PCR. The reaction
Ramakrishnan, Fink, and Levine
366
products can be electrophoresed on 1% agarose gels, gels dried, and amount of
radtoacttvity per reactton product band quantitated as before If necessary, the
amounts of RNA can be adJusted

Acknowledgments
The authors wish to thank Guthua Jtang for help with the zn situ hybrtdtza-
tton experiments and histology. This work was supported by Publtc Health
from the National Institute of Allergy and Infec-
Service grants ROlAI8228
ttous Diseases and ROlAG09470 from the National Institute of Aging and by a
grant from the Veteran™s Admmrstration.

References
1. Rock, D L and Fraser, N W (1983) Detection of HSV-I genome m central
nervous system of latently infected mice. Nature (Lord) 302,523-525
2 Efstathiou, S , Minson, A C , Field, H J , Anderson, J R , and Wildy, P (1986)
Detection of herpes simplex vu-us-specific DNA sequences in latently infected
mace and in humans. J Vu-01 51,44&455.
3 Spivack, J. G and Fraser, N W. (1987) Detection of herpes simplex virus type 1
transcripts durmg latent mfectton m mice J Vzrol 61,3841-3847
4 Deatly, A. M , Sptvack, J G., Lava, E , O™Boyle, D R , II, and Fraser, N W
(1988) Latent herpes simplex vn-us type 1 transcripts m peripheral and central
nervous system ttssues of mice map to stmtlar regtons of the viral genome
J Vwol. 62,74%756
5. Stevens, J. G , Wagner, E. K., Devi-Rao, G. B., Cook, M. L , and Feldman, L T.
(1987) RNA complementary to a herpesvuus gene mRNA 1sprominent m latently
infected neurons. Sczence 235, 1056-1059
6 Lynas, C , Laycock, K A., Cook, S D., Hill, T. J , Blyth, W. A., and Mattland, N
J (1989) Detection of herpes simplex virus type 1 gene expression m latently and
productively infected mouse ganglia using the polymerase chain reaction. J Gen
Vlrol IO, 2345-2355
7 Sawtell, N M. and Thompson, R. L (1992. Herpes simplex vnus type 1 latency-
associated transcription umt promotes anatomical site-dependent establishment
and reactivatton from latency J. Vrrol 66,2 157-2 169
8 Ramakrishan, R , Fmk, D J., Jiang, G , Desai, P., Glorioso, J C., and Levine, M.
(1994) Competttive quantitative PCR analysis of herpes simplex vnus type 1 DNA
and latency-associated transcript RNA m latently Infected cells of the rat brain J
Vlrol 68,1864--l 873.
9. Ramakrishnan, R., Levme, M., and Fink, D. J. (1994) A PCR-based analysis of
herpes simplex virus type 1 latency m the rat trigemmal ganglion estabhshed with
a rtbonucleottde reductase deficient mutant J. Vzrol 68, 7083-7091
10 Katz, J P., Bodin, E T , and Coen, D M. (1990) Quantitative polymerase chain
reaction analysis of herpes simplex vtrus DNA in ganglia of mice infected with
replication-incompetent mutants. J Vu-01 64,4288-4295
11 Fink, D. J , Sternberg, L R., Weber, P. C., Mata, M., Goms, W. F , and Glorioso,
Analysis of HSV-DNA and RNA 367
J. C. (1992) Zp1vwo expressron of P-galactosidase in hippocampal neurons by
HSV-mediated gene transfer Hum Gene Ther 3, 11-19
12 Rodahl, E and Stevens, J. G (1992) Differential accumulatton of herpes stmplex
virus type 1 latency-associated transcrtpts m sensory and autonomtc ganglia.
Vzrology 189,385388.
13 Gressens, P. and Martin, J. R. (1994) In sztu polymerase chant reactton. locahza-
tion of HSV-2 DNA sequences m infections of the nervous system. J. Vzrol. Meth-
ods 46,61-83
14. Becker-Andre, M. and Hahlbrock, K. (1989). Absolute mRNA quantttation using
the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript
titration assay (PATTY). Nucleic Acids Res 17,9437-9446.
15 Gtlltland, G., Perrm, S., and Bunn, H F (1990) Competmve PCR for quantitatton
of mRNA, in PCR Protocols. A Gwde to Methods and Applwatlons (Innis, M. A ,
Gelfand, D H., Smnsky, J J., and White, T. J., eds ), Academic, NY, pp. 60-69.
16 Chelly, J., Kaplan, J C., Mane, P., Gautron, S , and Kahn, A (1988) Transcrtp-
tion of the dystrophin gene m human muscle and nonmuscle tissues Nature
(Lond) 333,858-860.
17 Frye, R A., Benz, C C., and Liu, E. (1989) Detection of amplified oncogenes by
differential polymerase chain reaction. Oncogene 4, 1153-l 157.
18. Noonan, K E., Beck, C., Holzmayer, T. A.,Chin, J. E., Wunder, J. S., Andruhs, I.
L , Gazder, A. F , Willman, C. L., Griffith, B , Von Hoff, D D., and Roninson, I
B. (1990) Quantitative analysis of MDRl (multtdrug resistance) gene expression
m human tumors by polymerase chain reactton. Proc Nat1 Acad. Scl USA 87,
716@-7164
19 Henrard, D R., Mehaffey, W F., and Allain, J. P. (1992) A sensttwe viral capture
assay for detection of plasma vnemia in HIV infected mdivtduals AIDS Res Hum
Retrovw 68,47-52.
20. Wang, M., Doyle, M. V., and Mark, D. F. (1989) Quantrtation of mRNA by the
polymerase chain reaction Proc. Natl. Acad Sci USA 86,97 17-972 1.
21. Sperison, P., Wang, S. M , Reichenbach, P., and Nabholz, M (1992) A PCR-
based assay for reporter gene expression PCR Methods Appllc. 1, 164-170.
22. Wagner, E. K., Devi-Rao, G., Feldman, L. T., Dobson, A. T., Zhang, Y.-F.,
Flanagan, W. M., and Stevens, J. G. (1988). Physical charactertzatron of the herpes
simplex vnus latency-associated transcrtpt in neurons. J Vzrol. 62, 11941202.
23. NUOVO,G. J. (1992) PCR In Sztu Hybrzdizatzon Protocols and Applications
Raven, NY
24. Wang, R. F., Cao, W. W., and Johnson, M. G. (1992) A simplified, single tube,
single buffer system for RNA-PCR. BloTechniques 12,702-704.
25
HSV Vectors for Gene Therapy
David C. Bloom


1. Introduction
A number of aspects of the natural biology of herpes simplex vtrus (HSV)
make it an attractive candidate for a vector to express foreign genes within the
nervous system. Some of the advantages of an HSV vector are*
1 Establishment of a hfe-long latent mfectlon withm peripheral and central ner-
vous system neurons (for a review, see ref 1);
2 Latent HSV genomes exist as multiple episomal copies/neuron and integration is
not known to occur (2), and
3. Nonreplicatmg HSV recombinants can estabhsh a latent infection effclently (3)
This last point is perhaps the most important in that it permits the construc-
tion of safe, attenuated vectors for humans. In addition, there are other biologi-
cal properties of HSV that enhance its suitability as a vector from a practical
standpoint. The virus 1seasy to manipulate in vitro, so that recombmants con-
taming foreign genes can be constructed rapidly and its genome can accept
large inserts of DNA, making feasible the constructton of vectors that express
multiple therapeutic genes. This potential has understandably generated a great
deal of interest in exploiting HSV as a vector (4) and, to date, a number of
recombmants have been generated expressing reporters such as P-galactost-
dase (54), as well as biologically relevant peptrdes such as glucouronidase
(9), tyrosme hydroxylase (IO), and nerve growth factor (NGF) (1 I, 12) These
vectors mclude both recombinant HSV as well as a derivative termed
“amplicons.” Here we will be discussing only the former.
Early viral constructs expressed their respective markers transiently at high
levels. However, the expression declined rapidly with ttme. Although for some
therapeutic applicatrons transient expression of a peptide within target neurons
may be sufficient, for most uses,long-term expression from the latent infection
From Methods tn Molecular Medione, Vol 10 Herpes Simplex Vtrus Protocols
Edited by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ


369
370 Bloom

is desirable. Stable expression of genes from the context of the latent viral
genome has proven the most difficult problem to solve m the development of
HSV vectors. Smce latent infections of HSV are characterized by the absence
of viral transcription, with the exception of the latency-associated transcript
(LAT) (13), the LAT promoter would be the ideal candidate for the expression
of foreign genes during latency. However, recombmant viruses contammg the
genes for nerve growth factor (NGF) and /3-galactosidase driven by the LAT
promoter, express P-galactosidase and NGF RNA at high levels initially, but
not during the latent infection (12). A weaker promoter downstream of the
LAT promoter (LAP2) has also been proved to be msufficient for long-term
expression. A number of cellular promoters, mcludmg those for neuronal
housekeepmg genes, have been evaluated for their ability to express genes long
term m the nervous system within the context of the HSV genome. These con-
structs have expressed reporter genes at high levels during the acute mfection
(for a period of 2-10 d postmfection), but the levels of expression drop off
dramatically followmg the latent mfection (Z4) The Moloney Murme Leuke-
mia Virus (MoMuLV) LTR has been demonstrated however to afford long-
term expression of /3-galactosidase (7,15) when m the context of the LAT
promoter (26) This combination of the LAT core promoter and the MoMuLV
LTR allows extended expression of transgenes at high levels within the sen-
sory neurons of the peripheral nervous system (Z 6), but only mmimal levels of
sustained expression within the CNS (2.5). Work is still under way by a number
of groups to determine the elements and structural features of the LAT pro-
moter that allow long-term expression. In addition, work is still under way to
increase the levels of expression withm the CNS.
The focus of this chapter will be on the exploitation of HSV as a vector for
long-term expression within the nervous system. It will be concentrating on
important design considerations of vectors for specific uses, as well as meth-
ods for evaluating expression within animals. Other aspects such as prepara-
tion and growth of viral stocks, as well as reverse transcriptase-polymerase
chain reaction (RT-PCR) quantitatron of RNA transcripts, are presented else-
where in this volume. Since much of the evaluation of HSV vectors will ulti-
mately be performed in the ammal, basic techniques for testing of the constructs
m viva are presented here Because the specific assays employed will vary
greatly depending on the particular application, techniques, such as immuno-
hlstochemistry or znsztuhybridization (ISH), will not be discussed here.
2. Materials
1 Modified Eagle™sMedium (MEM) (Life Sciences, Bethesda,MD)* supplemented
with 5% calf serum, 250 U penicillin, 250 pg/mL streptomycin, 2 5 pg/mL
amphotericmB, and 292 pg/mL L-glutamme/mL.
371
HS V Vectors
2 Rabbit skm, Rat-2 (tk-), or Vero cells (Amerrcan Type Culture Collection,
Rockville, MD).
3. Trypsin: 1.25 g trypsin dissolved m 50 mL dH,O at 37°C Add 0.5 g EDTA, 20 g NaCl,
1 g KCI, 2 5 g dextrose, 0 5 g penicillin, and 0.25 g streptomycm. Brmg volume up to
200 mL with dH,O Filter-sterilize and store at 4°C Dilute 1 10 for workmg stock
4 T75 flasks and 60-mm dishes
5 Sterile 5-mL falcon tubes
6. TNE. 10 mMTris (pH 7 4), 1 mA4EDTA, O.lMNaCl. Filter-stertlize and store at
room temperature
7 2X HEPES (for 100 mL)* 1.6 g NaCl, 74 mg KCl, 37 mg Na,HPO, 7H,O, 0 2 g
dextrose, 1 g HEPES (free acid), pH to 7.05. Filter-stenhze, aliquot, and store at-20°C
8. 2.5M CaCl,: Filter-sterilize and store at room temperature
9 1X TE. 10 mA4Tris-HCl, pH 8.0, 1 mMEDTA m dH,O Autoclave
10 10% Sodium dodecyl sulfate (SDS): 100 g m 900 mL dH,O dissolved by heatmg
to 68™C, pH to 7 2, and add water to 1 L
11. Pronase: 20 mg/mL in dH,O. Store at -20°C. Self-digest for 2 h at 37™C
12 Viral lysis buffer. 10 mMTris-HCl, pH 8.0, 10 mMEDTA, 0 25% NaDOC, 0 5%
NP-40
13. Ethidmm bromide: 10 mg/mL m dH,O
14 Tris-borate buffer 89 mMTr1.s base, 89 mMboric acid, 10 MEDTA (pH -8 3).
15 Seakem agarose: (FMC, Rockland, ME).
16 2XMEM
17. Sterile, plugged Pasteur prpets
18 96-Well dish
19 6X Loading dye 0.25% bromophenol blue, 0.25% xylene cyanol, 15% F1coll400.
20 Hybond-N nylon membrane (Amersham, Arlington Heights, IL).
2 1. Whatman 3MM chromatography paper
22. Dot-blot apparatus
23. 1ONNaOH
24. 2.5M Tris-HCl, pH 7 6.
25 20X SSPE. 3.6MNaC1,O 2MNaH2P04, O.O2MEDTA, pH 7.s7.5 with NaOH
Autoclave.
26. BLOTTO stock: 5% nonfat dry milk, 0.2% Antifoam A (Sigma, St. LOUIS, MO)
m water. Warm to 42°C and stir well to completely dissolve. May be stored for
several months at 4°C
27 DNA probe labeling kit (Boehringer-Mannheim, Indianapolis, IN).
28 Neutral red solution 3.330 g/L (Life Technologres, Grand Island, NY)
29. 40% Dextrose* 40% dextrose w/v in distilled water. Filter-sterilize, and store at 4°C
30 Equilibrated phenol* Melt molecular-grade phenol at 68°C and add IMTris-HCI,
pH 8 0. Extract 2X with Tris-HCl, pH 8.0; 0.2% P-mercaptoethanol.
3 1. SEVAG. 24: 1 chloroformisoamyl alcohol.
32. 10% Saline: 10 g/l 00 mL dH*O. Filter-sterilize
33. Sodium pentobarbital: Nembutal (Abbott Laboratories, Abbott Park, IL) 7.5 mg/mL
m sterrle dH,O
Bloom
372
34 Dissection microscope
35 3c Dumont forceps
36 2X overlay agarose. 0 8% SeaKem ME agarose in dHzO. Autoclave
37 Dissecting scissors
38. Fme dissecting scissors
39. Microdissecting scissors
40 Solution A 0. IM NaOH, 1 5M NaCl
41 Solution B* 0.2M Tris-HCl, pH 7 6
42. Solution C 2X SSPE

3. Methods
3.7. Vector Design
Initial considerations m the design of HSV vectors depend largely on whether
the vector™s intended use is for cell culture, animal, or human use. The applica-
tton wtll dictate the degree of attenuation and duration of the transgene expres-
sion required. This section will discuss these spectlic points and then provide a
basic protocol for general constructton. It should be pointed out that once the
design is made, vector construction 1svery straightforward and can be performed
by anyone tramed in basic molecular techniques. Vector construction involves:
1. Construction of a recombmatton plasmid contammg the gene one wishes to
express behind a promoter. This plasmid contains recombmatton “arms” to allow
msertion of the promoter/gene construct mto the viral genome by homologous
recombmation,
2 Transfection of the plasmtd and viral DNA in cell culture,
3. Plaquing of the progeny, which will contain a mixture of wild type vnus and
recombinants, and
4 Screening for, and plaque purtfication of, the recombinants.

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