Journal of VjroZogicuf method, 35 (1991) 137-141 0 1991 Etsevier Science Publishers B.V. Al1 rights reserved. / 016~~34/91/$03.~
137
VIRMET 01243
Direct identification of recombinant vaccinia virus plaques by PCR L. Pasamontes’, IF. Hoffmann-La
J. Gubser2, R. Wittek2 and G.J. Viljoen2*
Roche Ltd., VFRM, Basel, Switzerland and 21nstitut de Biologic Animale, University of Luusanne. Lausanne, Switzerland (Accepted
IS June 1991)
Summary
A fast method for the identification of recombinant vaccinia viruses directly from individual plaques is described. Plaques are picked, resuspended in PBS-A and processed for PCR using two ‘uftiversal’ primers. The amplified sequences are analyzed by agarose gel el~tropho~s~s. This procedure allows discrimination between spontaneously arising TK-negative mutants, which do not carry the inserted gene, and the desired TK-negative recombinants resulting from insertional inactivation of the TK gene. Recombinant virus; Vaccinia; PCR; Plaque
Introduction
The first vaccinia virus recombinants were reported 10 years ago (Panicali and Paoletti, 1982; Mackett et al., 1982) and the use of this vector to express foreign genes has since steadily increased. The advantages of vaccinia virus as a vector include the relative ease with which recombinants can be generated, the high expression levels and correct processing of foreign proteins, as well as the large range of susceptible host cells. The main interest in recombinant vaccinia viruses today is their use as vaccine vectors, as it could be shown in a large number of different animal model systems that recombinant vaccinia viruses expressing genes from pathogens protected the animals against challenge with the pathogenic agent (Bennink and Yewdell, 1990). *Present address: Biochemistry Section, Onderstepoort, 0110, Republic of South Africa. Corre~~~~e~ce to: L. Pasamontes, F. Hoffmann-~ Roche Ltd., VFRM, 4002 Basel, Switzerland.
138
Several powerful methods have been developed to enrich the small percentage of recombinant virus obtained after homologous in vivo recombination (Miner and Hruby, 1990). In most cases, the foreign gene is inserted into the thymidine kinase (TK) gene and virus recombinants are selected for on the basis of their TK-negative phenotype. However, one ultimately has to distinguish between spontaneously arising TK-negative mutants and the desired TK-negative recombinants resulting from insertional inactivation of the TK gene. This can be accomplished by analysis of DNA of putative recombinants using different methods, e.g. restriction endonuclease fragment analysis, Southern blot, DNA dot-blot hybridization or by immunological means to detect the foreign gene product, e.g. immunoprecipitation, immunofluorescence, immuno dot-blot and Western blot. All these methods are labour-intensive, time-consuming and, as usually several plaque purifications are necessary to ensure a pure preparation of recombinant virus, considerable time can be saved using the method described below.
Materials and Methods Considering that most laboratories use one plasmid vector to target the different genes into the vaccinia virus genome, we designed a protocol to identify different recombinant viruses using the polymerase chain reaction (PCR) and two ‘universal’ primers to amplify any given sequence introduced into the cloning site. Fig. 1 shows the relevant portions of three recombinant plasmids used to generate recombinant virus. They contain foreign gene sequences of different lengths fused to the vaccinia virus 7.5kDa gene promoter. These chimeric genes are flanked by viral TK sequences to allow homologous recombination. 530 bp I
IA 1+
TK
p
P7.5 B 702 bp I
!L 2-l
TK
H
~7.5
H
MAE-1
HTKj B 1525 bp
I
IA
-
3-
L
R3
H
TK
j-
B
Fig. 1. (1) Recombination plasmid pMABI-2; (2) recombination plasmid pMAB-1; (3) recombination plasmid R3. A and B show the localization of the primers used for PCR amplification. The sizes of the expected PCR amplification products are indicated.
139
Fig. 2. PCR-amplified plaques from recombinant virus obtained with plasmid pMABI-2. Four plaques (lanes 2-5) picked randomly were PCR-amplified using primer A: S-ATTGCACGGTAAGGAAGTAG-3 and B: 5’-GTCCCATCGAGTGCGGCTAC-3’. 10 ~1 of the PCR reaction mixture was electrophoresed on a 1.5% agarose gel. The expected size of the amplified fragment for recombinant viruses carrying the gene MABl-2 is 530 bp. Lanes 1 and 6: DRIgestTM III.
The construction of recombinant vaccinia viruses was carried out according to previously described methods (Drillien and Spehner, 1983; Mackett et al., 1985). Briefly, CVl cell cultures were infected with the temperature-sensitive vaccinia virus ts7 (Drillien and Spehner, 1983) for 3 h at the permissive temperature of 33°C. Calcium-phosphate-precipitated DNA, typically 100-500 ng of plasmid DNA and 200 ng of wild-type vaccinia virus DNA, was then deposited on the cells and left for 1 h at room temperature. The cell cultures were incubated at 395°C for 2 h before a glycerol treatment was applied. Virus was allowed to grow for two days at 39.5”C. The cells were then subjected to three freeze/thaw cycles to liberate the virions. Putative recombinants were isolated by plaque purification on the basis of their TK- phenotype using human TK- 143B cells and bromdesoxyuridin as selective agent. Virus plaques were picked using a Pasteur pipet and resuspended in 100 ~1 of PBS-A, freeze/ thawed 3 times and sonitied for 30 s in a sonic waterbath. One half of this material was stored at -70°C for subsequent virus amplification. The other half was phenol/chloroform extracted and precipitated with ethanol in the presence of 10 ,ug yeast tRNA. The precipitates were washed once with 70% ethanol before drying. The DNA was dissolved in Hz0 and used for PCR amplification (100 ~1 PCR reaction mixture consisting of: 2.5 ng of each primer A and B (see Figs. 1 and 2), 16.6 mM (NH&S04, 67 mM Tris-HCl (pH 8.8), 6.7 mM MgC12, 10 mM 2-mercaptoethanol and 0.2 mg/ml bovine serum albumin, 200 PM of each dNTP and 1 U Taq polymerase). To ensure that the vaccinia DNA was completely denatured, two cycles of heating at 95°C for 5 min and cooling to 55°C for 1 min were introduced before a 30-cycle PCR amplification run was performed. It consisted of: denaturing the DNA at 95°C for 1 min, annealing of the primers at 55°C for 2 min and elongation of the primers at 72°C for 3 min. After PCR amplification, 10 ~1 of the reaction
140
Fig. 3. PCR amplification of plaques obtained from three different recombinant vaccinia viruses. One plaque of each vaccinia virus recombinant derived from plasmids pMABI-2 (lane 3), pMAB1 (lane 4) R3 (lane 5) and a control plaque from wild-type vaccinia virus (lane 2) were PCR-amplified and separated on a 1.5% agarose gel. The expected band sizes are 530 bp for pMABI- 2,702 bp for pMAB1 and 1525 bp for R3. No amplification was detected using the wild-type vaccinia virus plaque. Lanes 1and 6: DRIgestTM III Marker (Pha~acia).
mixture was electrophoresed on a 1.5% agarose gel and stained with ethidium bromide.
Results and Discussion Fig. 2 shows the result of four randomly picked plaques from recombinant virus derived from plasmid pMABl-2. All four viruses analyzed contain the expected insert of 530 bp. Fig. 3 shows the results obtained from individual plaques of three different recombinant viruses carrying inserts of 530, 702, and 1525 bp derived from plasmids pMABl -2, pMAB-1 and R3, respectively. If further characterization of the recombinants is required, the PCR-amplified DNA may be analyzed by restriction enzymes or Southern blot. Finally, this method may be adapted to any given vaccinia virus promoter by replacing the primer A (Fig. 1) with a primer hybridizing to the proximal region of the TK gene giving thus the possibility to amplify introduced sequences independently of the vaccinia virus promoter used. Although this procedure was developed for the identification of recombinant vaccinia viruses, it should be useful for identifying other recombinant viruses.
References Bennink, J.R. and Yewdell, J.W. (1990) Recombinant vaccinia viruses as vectors for studying T lymphocyte specificity and function. In: R.W. Moyer and PC. Tuner (Ed@, Poxviruses. Curr. Top. Microbial. Immunol. 163, 153-184.
141 Drillien, R. and Spehner, D. (1983) Physical mapping of vaccinia virus temperature sensitive mutations. Virology 131, 385-393. Mackett, M., Smith, G.L. and Moss, B. (1982) Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc. Natl. Acad. Sci. USA 79, 7415-7419. Mackett, M., Smith, G.L. and Moss, B. (1985) The construction and characterization of vaccinia virus recombinants expressing foreign genes. In: D.M. Glover (Ed), DNA Cloning-A Practical Approach. IRL Press, pp. 191-211. Miner, J.N. and Hruby, D.E. (1990) Vaccinia virus: a versatile tool for molecular biologists. TIBTECH 8, 20-25. Panicali, D. and Paoietti, E. (1982) Construction of poxviruses as cloning vectors: Insertion of the thymidine kinase gene from herpes simplex into the DNA of infectious vaccinia virus. Proc. Natl. Acad. Sci. USA 79, 49274931.
137
VIRMET 01243
Direct identification of recombinant vaccinia virus plaques by PCR L. Pasamontes’, IF. Hoffmann-La
J. Gubser2, R. Wittek2 and G.J. Viljoen2*
Roche Ltd., VFRM, Basel, Switzerland and 21nstitut de Biologic Animale, University of Luusanne. Lausanne, Switzerland (Accepted
IS June 1991)
Summary
A fast method for the identification of recombinant vaccinia viruses directly from individual plaques is described. Plaques are picked, resuspended in PBS-A and processed for PCR using two ‘uftiversal’ primers. The amplified sequences are analyzed by agarose gel el~tropho~s~s. This procedure allows discrimination between spontaneously arising TK-negative mutants, which do not carry the inserted gene, and the desired TK-negative recombinants resulting from insertional inactivation of the TK gene. Recombinant virus; Vaccinia; PCR; Plaque
Introduction
The first vaccinia virus recombinants were reported 10 years ago (Panicali and Paoletti, 1982; Mackett et al., 1982) and the use of this vector to express foreign genes has since steadily increased. The advantages of vaccinia virus as a vector include the relative ease with which recombinants can be generated, the high expression levels and correct processing of foreign proteins, as well as the large range of susceptible host cells. The main interest in recombinant vaccinia viruses today is their use as vaccine vectors, as it could be shown in a large number of different animal model systems that recombinant vaccinia viruses expressing genes from pathogens protected the animals against challenge with the pathogenic agent (Bennink and Yewdell, 1990). *Present address: Biochemistry Section, Onderstepoort, 0110, Republic of South Africa. Corre~~~~e~ce to: L. Pasamontes, F. Hoffmann-~ Roche Ltd., VFRM, 4002 Basel, Switzerland.
138
Several powerful methods have been developed to enrich the small percentage of recombinant virus obtained after homologous in vivo recombination (Miner and Hruby, 1990). In most cases, the foreign gene is inserted into the thymidine kinase (TK) gene and virus recombinants are selected for on the basis of their TK-negative phenotype. However, one ultimately has to distinguish between spontaneously arising TK-negative mutants and the desired TK-negative recombinants resulting from insertional inactivation of the TK gene. This can be accomplished by analysis of DNA of putative recombinants using different methods, e.g. restriction endonuclease fragment analysis, Southern blot, DNA dot-blot hybridization or by immunological means to detect the foreign gene product, e.g. immunoprecipitation, immunofluorescence, immuno dot-blot and Western blot. All these methods are labour-intensive, time-consuming and, as usually several plaque purifications are necessary to ensure a pure preparation of recombinant virus, considerable time can be saved using the method described below.
Materials and Methods Considering that most laboratories use one plasmid vector to target the different genes into the vaccinia virus genome, we designed a protocol to identify different recombinant viruses using the polymerase chain reaction (PCR) and two ‘universal’ primers to amplify any given sequence introduced into the cloning site. Fig. 1 shows the relevant portions of three recombinant plasmids used to generate recombinant virus. They contain foreign gene sequences of different lengths fused to the vaccinia virus 7.5kDa gene promoter. These chimeric genes are flanked by viral TK sequences to allow homologous recombination. 530 bp I
IA 1+
TK
p
P7.5 B 702 bp I
!L 2-l
TK
H
~7.5
H
MAE-1
HTKj B 1525 bp
I
IA
-
3-
L
R3
H
TK
j-
B
Fig. 1. (1) Recombination plasmid pMABI-2; (2) recombination plasmid pMAB-1; (3) recombination plasmid R3. A and B show the localization of the primers used for PCR amplification. The sizes of the expected PCR amplification products are indicated.
139
Fig. 2. PCR-amplified plaques from recombinant virus obtained with plasmid pMABI-2. Four plaques (lanes 2-5) picked randomly were PCR-amplified using primer A: S-ATTGCACGGTAAGGAAGTAG-3 and B: 5’-GTCCCATCGAGTGCGGCTAC-3’. 10 ~1 of the PCR reaction mixture was electrophoresed on a 1.5% agarose gel. The expected size of the amplified fragment for recombinant viruses carrying the gene MABl-2 is 530 bp. Lanes 1 and 6: DRIgestTM III.
The construction of recombinant vaccinia viruses was carried out according to previously described methods (Drillien and Spehner, 1983; Mackett et al., 1985). Briefly, CVl cell cultures were infected with the temperature-sensitive vaccinia virus ts7 (Drillien and Spehner, 1983) for 3 h at the permissive temperature of 33°C. Calcium-phosphate-precipitated DNA, typically 100-500 ng of plasmid DNA and 200 ng of wild-type vaccinia virus DNA, was then deposited on the cells and left for 1 h at room temperature. The cell cultures were incubated at 395°C for 2 h before a glycerol treatment was applied. Virus was allowed to grow for two days at 39.5”C. The cells were then subjected to three freeze/thaw cycles to liberate the virions. Putative recombinants were isolated by plaque purification on the basis of their TK- phenotype using human TK- 143B cells and bromdesoxyuridin as selective agent. Virus plaques were picked using a Pasteur pipet and resuspended in 100 ~1 of PBS-A, freeze/ thawed 3 times and sonitied for 30 s in a sonic waterbath. One half of this material was stored at -70°C for subsequent virus amplification. The other half was phenol/chloroform extracted and precipitated with ethanol in the presence of 10 ,ug yeast tRNA. The precipitates were washed once with 70% ethanol before drying. The DNA was dissolved in Hz0 and used for PCR amplification (100 ~1 PCR reaction mixture consisting of: 2.5 ng of each primer A and B (see Figs. 1 and 2), 16.6 mM (NH&S04, 67 mM Tris-HCl (pH 8.8), 6.7 mM MgC12, 10 mM 2-mercaptoethanol and 0.2 mg/ml bovine serum albumin, 200 PM of each dNTP and 1 U Taq polymerase). To ensure that the vaccinia DNA was completely denatured, two cycles of heating at 95°C for 5 min and cooling to 55°C for 1 min were introduced before a 30-cycle PCR amplification run was performed. It consisted of: denaturing the DNA at 95°C for 1 min, annealing of the primers at 55°C for 2 min and elongation of the primers at 72°C for 3 min. After PCR amplification, 10 ~1 of the reaction
140
Fig. 3. PCR amplification of plaques obtained from three different recombinant vaccinia viruses. One plaque of each vaccinia virus recombinant derived from plasmids pMABI-2 (lane 3), pMAB1 (lane 4) R3 (lane 5) and a control plaque from wild-type vaccinia virus (lane 2) were PCR-amplified and separated on a 1.5% agarose gel. The expected band sizes are 530 bp for pMABI- 2,702 bp for pMAB1 and 1525 bp for R3. No amplification was detected using the wild-type vaccinia virus plaque. Lanes 1and 6: DRIgestTM III Marker (Pha~acia).
mixture was electrophoresed on a 1.5% agarose gel and stained with ethidium bromide.
Results and Discussion Fig. 2 shows the result of four randomly picked plaques from recombinant virus derived from plasmid pMABl-2. All four viruses analyzed contain the expected insert of 530 bp. Fig. 3 shows the results obtained from individual plaques of three different recombinant viruses carrying inserts of 530, 702, and 1525 bp derived from plasmids pMABl -2, pMAB-1 and R3, respectively. If further characterization of the recombinants is required, the PCR-amplified DNA may be analyzed by restriction enzymes or Southern blot. Finally, this method may be adapted to any given vaccinia virus promoter by replacing the primer A (Fig. 1) with a primer hybridizing to the proximal region of the TK gene giving thus the possibility to amplify introduced sequences independently of the vaccinia virus promoter used. Although this procedure was developed for the identification of recombinant vaccinia viruses, it should be useful for identifying other recombinant viruses.
References Bennink, J.R. and Yewdell, J.W. (1990) Recombinant vaccinia viruses as vectors for studying T lymphocyte specificity and function. In: R.W. Moyer and PC. Tuner (Ed@, Poxviruses. Curr. Top. Microbial. Immunol. 163, 153-184.
141 Drillien, R. and Spehner, D. (1983) Physical mapping of vaccinia virus temperature sensitive mutations. Virology 131, 385-393. Mackett, M., Smith, G.L. and Moss, B. (1982) Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc. Natl. Acad. Sci. USA 79, 7415-7419. Mackett, M., Smith, G.L. and Moss, B. (1985) The construction and characterization of vaccinia virus recombinants expressing foreign genes. In: D.M. Glover (Ed), DNA Cloning-A Practical Approach. IRL Press, pp. 191-211. Miner, J.N. and Hruby, D.E. (1990) Vaccinia virus: a versatile tool for molecular biologists. TIBTECH 8, 20-25. Panicali, D. and Paoietti, E. (1982) Construction of poxviruses as cloning vectors: Insertion of the thymidine kinase gene from herpes simplex into the DNA of infectious vaccinia virus. Proc. Natl. Acad. Sci. USA 79, 49274931.
