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39 This ˜111 inactivate the enzyme and also favor the formation of double-stranded
ohgonucleottdes over self-annealed oligonucleottdes
40 If the sites for two diagnostic enzymes have been mapped, the site of insertion of
the mutagenic oltgonucleotlde may also (and possibly more precisely) be deter-
mmed by two double digestions, each containing a different diagnostic enzyme
and the enzyme that cleaves the mutagenic ohgonucleottde.
41 The transformation efficiency of ultracompetent cells drops when more than
20-50 ng DNA are added per transformation
42 We continue analyzing mutant cosmid derivatives until we have a density of
approxnnately one ohgonucleotide msertton per 250-500 bp
43 To generate double (or triple or quadruple) mutants, one may (m a later stage)
also Include mutant dertvattves of more than one cosmrd clone m an overlap
recombmation
44 The proof that a spectfic gene is essential (or that an observed phenotype can be
attributed to the inserted oligonucleotide) can be given m two ways: (1) by
cotransfectmg a wild-type cloned DNA fragment overlapping the inserted ohgo-
nucleottde (13) The oligonucleotlde will be replaced, by homologous recombi-
nation, with the wild-type fragment This should result m the generation of
vtable, wild-type vnus and (2) by constructing a cell lme that provides the wild
type gene in tram by stable transfectton (refs 9 and 10, and Chapter 6 of this
volume). This should now allow the ohgonucleottde-contammg mutant vtrus to
be viable after overlap recombmation
45 It is advised not to discard the resulting gel but to blot it on a mtrocellulose or
nylon membrane (see elsewhere m of this volume for a protocol on Southern
blotting) The resulting blot may later be used to characterize small subclones
that flank the inserted oligonucleotides (as described m Fig. 1C and Section 3 3 )
46 Of the cloned virus fragments to be cotransfected here, the nonmutagemzed frag-
ments are CsCl gradient purified cloned fragments, liberated from the vector (see
Section 3.1.15 ), the fourth fragment is the gelpurified mutant insert fragment In
total, every transfectton consumes 2 mg DNA, each of the four (or five) frag-
ments amounting equimolarly
47. If no viable virus is generated after overlap recombmation including a mutant
cosmid fragment, repeat the overlap recombmation twice to ascertam that the
failure of overlap recombmation IS not owmg to failure of the transfection itself
Based on our results with PRV (5,6) and on mutagenesis studies with HSV-1
(reviewed in ref 23), about half of the overlap recombmations are expected to
yield viable mutant virus
48. Since the PRV genome has a G.C content of 73%, Sau3A sites occur less than
once m every 256 bp. Consequently, most flankmg probes are larger than 200 bp.
For another herpesvirus, average insert sizes may be smaller or larger, depending
on the G C content of the vnus.
49. Any high copynumber vector that contains a polylinker, e.g., one from the pUC,
85
Characterization of a Herpesvirus Genome
the pSP, the pGEM, or the pBluescript series, may be used here Vectors con-
taming a single-strand replication origin may be preferred if the inserts are to
be sequenced.
If no compatible site is found m the polylinker of the vector, the vector may be
50
cleaved with both BumHI and an enzyme that leaves a blunt end (e.g , EcoRV)
The ohgonucleotide-containing cosmid derivatives are m the latter case first
digested with the ohgonucleotide cleaving enzyme, blunted with the Klenow
enzyme, or with T4 DNA polymerase (see Section 3.1.5.) and next with Suu3A.
51 A light molar excess of vector over insert fragments (note that the insert frag-
ments ˜111,on the average, have a size of a mere 128 bp if the virus genome has
a GC content of 50%) and a large volume of the ligation will promote the cloning
of a single fragment only.
We use an MspI digest of pSP72, yielding fragments of 501,430,404,242,237,
52
190, 147, 110,67,40,34 (2X), and 26 bp, in addition to a lambda HzndIII digest.
53. If one of the flanking clones is too small, it may not be detected on the gel. This
problem may be circumvented by digesting more DNA, and mcreasmg the agar-
ose percentage of the gel to 3 or 4%. Alternatively, one may be content with only
a single flanking probe, or use another four-cuttmg enzyme than Sau3A for the
cloning of the flanking probes.
54 SspI does not cut these HSV- 1 fragments but, instead, the vector only Since, for
technical reasons, the HSV- 1 inserts could not be cut out of the vector, this was
done to physically separate the HSV-1 DNA fragments that are present at either
side of the vector
55 Most PRV flanking probes have sizes greater than 200 bp Smaller probes are
labeled mefficiently by random priming. Therefore, concatemerization of very
small probes by ligation is advised to improve labeling efficiency.

References
1 Karlm, S , Mocarski, E. S., and Schachtel, G. A. (1994) Molecular evolution of
herpesviruses: genomic and protein sequence comparisons. J Vtrol. 68,1886-l 902.
2 van Zijl, M , Quint, W., Briaire, J., de Rover, T., Gielkens, A., and Berns, A
(1988) Regeneration of herpesvtruses from molecularly cloned subgenomic frag-
ments. J VlroZ 62,2191-2195.
3 Bernards, R., Vaessen, M. J., Van der Eb, A. J., and Sussenbach, J S (1983)
Construction and characterrzatton of an adenovirus type S/adenovirus type 12
recombinant virus. Vzrology 13,30-38.
4. Kapoor, Q. S. and Chinnadurai, G. (1981) Method for introducing site-specific
mutations into adenovnus 2 genome: construction of a small deletion mutant in
VA-RNA1 gene Proc Nat1 Acad. Scz USA 78,2184-2188
5 de Wind, N., Zijderveld, A., Glazenburg, K., Gielkens, A., and Berns, A. (1990)
Linker insertion mutagenesis of herpesviruses: mactivation of single genes withm
the unique short region of pseudorabies w-us. J Vim1 64,46914696.
6. de Wind, N , Peeters, B. P H., Zijderveld, A., Gielkens, A. L. J., Berns, A J. M ,
and Kmrman, T. G. (1994) Mutagenesis and characterization of a 41 kilobase pair
86 de Wind, van Zul, and Berm
region of the pseudorabies vu-us genome: transcription map, search for virulence
genes, and comparison with homologs of herpes simplex vu-us type 1 Vzrology
200,784-790
7. Parker, R. C. (1980) Conversron of circular DNA to linear strands for mapping.
Meth Enzymol 65,4 15-426
8. van Zijl, M., van der Gulden, H , de Wind, N , Grelkens, A , and Berm, A (1990)
Identificatron of two genes m the untque short region of pseudorabres virus; com-
parison with herpes simplex virus and varicella-zoster virus. J Gen Vwol 71,
1747-1755.
9 Peeters, B., de Wind, N., Hootsma, M., Wagenaar, F., Gielkens, A., and
Moormann, R (1992) Pseudorabies vnus envelope glycoprotems gp50 and gI1
are essential for vn-us penetration but only gII ts mvolved m membrane fusion J
Vu-01 66, 894-905
Peeters, I3 , de Wmd, N , Broer, R., Gielkens, A., and Moormann, R (1992) Gly-
10
coprotein gH of pseudorabies virus is essential for entry and cell-to-cell spread of
the virus J Vu-01 66,3888-3892
11. de Wind, N , Domen, J., and Berns, A (1992) Herpes viruses encode an unusual
protein-serine/threonme kmase which is nonessential for growth m cultured cells
J Vzrol 66,5200-5209
12. de Wind, N , Wagenaar, F , Pol, J , Kimman, T , and Berns, A. (1992) The
pseudorabies virus homolog of the herpes simplex virus UL21 gene is a capsid
protein which is mvolved m capsid maturation J Vzrol 66, 7096-7103
13 de Wmd, N , Gielkens, A., Berns, A., and Krmman, T. (1993) Ribonucleotide
reductase-deficient mutants ofpseudorabres virus are avirulent for pigs and Induce
partial protective rmmumty J Gen Vu-01 74, 35 l-359.
14 Wagenaar, F , Pol, J. M. A , Peeters, B , Gtelkens, A. L J , de Wind, N , and
Knnman, T G (1995) The US3-encoded potem kmase from pseudorabies virus
affects egress of virrons from the nucleus J Gen Vzrol 76, 185 l-1 859
15. Kmnnan, T G , de Wind, N., Oei-Lie, N , Pol, J. M. A., Berns, A. J M., and
Gielkens, A L J (1992) Contribution of single genes within the Us region of
AuJeszky™s disease vnus (suid herpes virus type 1) to virulence, pathogenesis and
unmunogentcity J. Gen Vlrol 73, 243-251
16 Kimman, T. G., de Wind, N., de Bruin, T., de Visser, Y , and Voermans, J (1994)
Inactivatton of glycoprotem gE and thymidme kmase synergistically decreases m
VIVO replicatton of pseudorabres virus and the mduction of protective immunity
Vzrology 205,5 1I-5 18.
17. Alexander, D. C. (1987) An efficient vector-primer cDNA cloning system Meth-
ods Enzymol. 154,4 l-64
18. Bnnboim, H. C and Daly, J. (1979) A rapid alkalme extraction procedure for
screenmg recombinant plasmid DNA. Nuclezc Acids Res 7, 15 13-1523
19. Graham, F. L. and Van der Eb, A J. (1973) Transformation of rat cells by DNA of
human adenovrrus 5. Vwology 54,536-539.
20. Hanahan, D. (1985) Techniques for transformation of E colz , m DNA Cloning, a
Pructzcal Approach, vol. 1. (Glover, D. M., ed ), IRL, Oxford, pp 109-135
87
Characterization of a Herpesvws Genome
21. McGeoch, D. J., Dalrymple, M. A., Davison, A J., Dolan, A., Frame, M C ,
McNab, D , Perry, L. J , Scott, J E , and Taylor, P. (1988) The complete DNA
sequence of the long unique region in the genome of herpes simplex vnus type 1
J Gen. Vwol. 69, 1531-1574
22 Hooft van Iddekinge, B J L , de Wind, N , Wensvoort, G., Kimman, T G.,
Gielkens. A. L J , and Moormann, R. J M. (1997) Comparison of the protective
efficacy of new btvalent recommant live vaccmes against pseudorabres and clas-
sical swine fever m prgs, in preparatron
23 Ward, P. L and Rotzman, B. (1994) Herpes szmplex genes the blueprmt of a
successful human pathogen Trends Genet 10, 167-274
6
Construction and Use of Cell Lines
Expressing HSV Genes
Claire Entwisle

1. Introduction
Complementary cell lines have become an accepted tool for the func-
tional analysis of viral genes (1,2) and proteins, as well as an essential com-
ponent m strategies for the construction of mutant viruses. More recent
applications include the propagation of repllcatlon-defective virus prod-
ucts with potential as viral vaccines (3,4) or as vehicles for gene therapy
(.5,6). The perceived requirements for such systems are low recombinatton
frequencies between complementmg cell and recombinant virus, stable ex-
pression of the complementmg protein withm the cell, and efficient comple-
mentation. The standard techniques of eukaryotlc cell transfectlon and
clonal selection are routinely employed m the generation of complemen-
tary cell lines, and are described briefly in this chapter. Perhaps a more
novel mtroduction to this field is the possibility of using transgemc tech-
nology. Transgemc ammals have the potential to provide both an in vivo
model of complementation (7) and a comprehensive library of novel
complementing cell types, particularly cell types resistant to traditional
transfection protocols.
1.1. Principle
The introduction of plasmid DNA into eukaryotic cells can be accomplished
by several techniques: calcium chloride precipitation, dextran, electroporation,
or liposome fusion (8). Each method has particular advantages and dlsadvan-
tages, and may be more or less suitable for a particular cell type. Series of trial
transfectlons using a test vector and reporter gene can be set up to determine
appropriate transfection conditions (8). LipofectinTM reagent (Gibco-BRL) 1s
From Methods IR Molecular Medxme, Vol 10 Herpes Smplex Wrus Protocols
EdRed by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ

89
Entwisle
90




ampR SV40 Promoter




Fig. 1. Expression vector. Unique sites in polylinker HindIII, &XI, NotI, XbaI,
and ApaI.


generally applicable to most cell types. Methods and recommended uses are
supplied by the manufacturer. Calcium chloride precipitation is a simple
alternative, and requires no investment in expensive equipment or spe-
cialized reagents. A general method is described in this chapter.
Several commercially available plasmids are suitable for mammalian
cell expression (Fig. 1). Suppliers are listed Section 2. Expression vec-
tors generally include a selection cassette, consisting of a constitutive
promoter driving a selection marker, an amp resistance marker for plas-
mid maintenance in Escherichia coli, and an expression cassette with a
promoter sequence, insertion linker, and poly A signal.
When selecting or modifying an available plasmid, it is important to
consider the activity and source of the expression promoter, whether to
choose a constitutive (I) or inducible system, and whether early or late
expression within the viral replication cycle is a particular requirement.
Viral specific inducible promoters (4) are particularly useful for the ex-
pression of proteins toxic to cell survival. Even with proteins considered
nontoxic, expression from an inducible promoter may increase cell line
stability. The homologous promoter and poly A signals should also be
considered and may be appropriate for certain experimental protocols.
Unfortunately, this approach can sometimes lead to later problems with
unacceptable levels of recombination between the cell line and deleted virus.
There are a number of selection systems available. The most widely used
dominant selection system is the neoresistance marker, which introduces resist-
ance to the antibiotic Geneticin @ Other useful systems are puromycin,
.
hygromycin, HSV tk, and His D. The particular choice of system will depend
on the pre-existing phenotype of the target cell type and the availability of the
respective antibiotic/or marker plasmid.
Cell lines Expressing HSV Genes 91

2. Materials
2.1. CaCl, Transfection of Vero Cells
1. Dulbecco™s modified Eagle™s medium (DMEM)
2. Fetal calf serum
3. Phosphate-buffered salme, magnesium-, and calcium-free.
4 1X Trypsin-EDTA, ICN Blomedlcals, Inc , cat no 16-891-49.
5 2X HBS* 280 mA4 NaCl, 50 mM HEPES, 1 5 mM Na2HP04, m HZ0 Adjust to
pH 7.1-7 2. Sterilize the stock solution by filtration, and store at -20°C m 20-mL
aliquots
6 TE buffer 10 mM Tns-HCl, pH 7.5, 1 mM EDTA, and filter-sterihze
7 Ethanol
8 3M sodium acetate, pH 5 0.
9. Stock solution of G4 18 (Sigma, St. Loius, MO, Geneticin@ product no. G95 16)
G418 is dissolved m to give a stock solution at 10 mg/mL. Filter sterlhze, and
store aliquots at -20°C
10. Vero cell growth media (GM). DMEM, 10% FCS, 1% glutamme. Transfectants
are selected at a concentration of 600 pg/mL G4 18 m GM
11. Tissue-culture grade flasks and Petri dashes.
12 Mammalian expresslon plasmlds are avallable from Invltrogen (R&D Systems,
UK), Promega Corporation, and Clontech (Cambridge Bioscience, UK)
13 The pCAT and Luclferase reporter gene systems are avallable from Promega
(Southampton, UK)
2.2. Screening of Cell Lines by PCR
1. Digestion buffer 5 mMEDTA, pH 8.0,200 mMNaC1, 100 mMTns-Hcl, pH 8.0,
0.2% SDS
2 Proteinase K (Sigma cat no P2308) 10 mg/mL m UHP H20.
3. 10X PCR buffer. 100 mA4 Tris-HCl, 15 mMMgC12, 500 mA4 KCI, pH 8 3
4 Equilibrated phenol/chloroform* 50/50 mix of Tns-saturated phenol, pH 8.0
(FSA, laboratory supplies), and chloroform
5. Tuq DNA polymerase (5 U/mL Boehrmger).
6 dNTPmlx.
1.25 mM of each dNTP diluted mto UHP H20
100 mM stocks are available from Boehringer
dATP lithium salt 100 mM (cat, no. 105 1440)
dCTP lithium salt 100 mM (cat. no. 105 1458).
dGTP lithium salt 100 mM (cat. no 105 1466).
dTTP lithium salt 100 mM(cat. no. 1051482)
7 Light mineral oil (Sigma cat. no. M5904)
8. PCR primers diluted to 50 pmol/pL. For PCR from genomlc templates, 30-mer
oligos are recommended For RT-PCR, use 18-22 mer wlthm the coding sequence
9. 1 L TBE buffer (10X) 108 g Tris base, 55 g boric acid, 9.3 g EDTA It should be
unnecessary to adjust the pH of this solution However, check that the pH IS 8 2-8 5
Entwisle
92

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