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Characterization of Recombinant Vaccinia Viruses and Their Products

Patricia L. Earl¹, Bernard Moss¹

¹National Institute of Allergy and Infectious Diseases, Bethesda, Maryland

Publication Name:

Current Protocols in Molecular Biology

Unit Number:

Unit 16.18

DOI:

10.1002/0471142727.mb1618s43

Online Posting Date:

May, 2001

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Abstract

After a recombinant vaccinia virus is made, its DNA and protein products can be analyzed in several ways. Protocols are provided in this unit for identification of the recombinant virus by PCR (with verification of correct insertion of the DNA by Southern blotting) and by dot-blot hybridization. Also, when antibodies are available, protein expression can be analyzed by immunological methods detailed here such as dot blotting with an antibody, immunoblotting and/or immunoprecipitation. In addition, immunostaining can be used for identification of recombinant plaques as well as for determination of the purity of a recombinant virus stock. All of the protocols in this unit can be used for characterization of modified vaccinia virus Ankara (MVA) recombinant viruses.

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Unit Introduction
Basic Protocol 1: Detection of Vaccinia DNA Using PCR
Basic Protocol 2: Detection of Vaccinia DNA Using Southern Blot Hybridization
Basic Protocol 3: Detection of Vaccinia DNA Using Dot-Blot Hybridization
Alternate Protocol: Detection of Expressed Protein by a Dot-Blot Procedure
Basic Protocol 4: Detection of Expressed Protein Using Immunoblotting
Basic Protocol 5: Detection of Expressed Protein Using Immunoprecipitation
Reagents and Solutions
Commentary
Literature Cited

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Materials

Basic Protocol 1: Detection of Vaccinia DNA Using PCR

Materials

Confluent BS-C-1 or HuTK^– 143B cell monolayer (unit 16.16)
Phosphate-buffered saline (PBS; appendix 2)
Trypsin/EDTA (0.25%:0.02%), 37°C
Complete MEM-10, -2.5, and -5 media (unit 16.16)
Complete MEM-10/BrdU medium (unit 16.16)
Recombinant virus plaques, picked and resuspended in tubes (unit 16.17)
Selective agents (filter sterilize and store at –20°C; also see unit 16.17):
10 mg/ml (400×) mycophenolic acid (MPA; Calbiochem) in 0.1 N NaOH (for XGPRT selection)
10 mg/ml (40×) xanthine in 0.1 N NaOH (for XGPRT selection)
10 mg/ml (670×) hypoxanthine in 0.1 N NaOH (for XGPRT selection)
5 mg/ml (200×) 5-bromodeoxyuridine (BrdU) in water (for TK selection)
Dry ice/ethanol bath
DNA extraction buffer (see recipe)
3 M sodium acetate, pH 6.0 (appendix 2)
95% and 70% ethanol, ice-cold
10× MgCl₂-free PCR amplification buffer (unit 15.1)
10 mM 4dNTP mix (unit 15.1)
100 µM primer (sequence depends on where foreign gene is inserted; see unit 2.11 for oligonucleotide synthesis strategies)
15 mM MgCl₂
24-well tissue culture plates
Cup sonicator
Additional reagents and equipment for tissue culture and counting cells (appendix 3F), phenol/chloroform extraction of DNA, PCR amplification (unit 15.1), and agarose gel electrophoresis (unit 2.5)

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 2: Detection of Vaccinia DNA Using Southern Blot Hybridization

Materials

Confluent BS-C-1 cell monolayer (unit 16.16)
Complete complete MEM-10 and -5 media (unit 16.16)
Recombinant vaccinia virus stock (unit 16.16)
0.25 mg/ml trypsin (2× crystallized and salt-free;Worthington; filter sterilize and store at –20°C)
Phosphate-buffered saline (PBS; appendix 2)
Low-salt buffer (see recipe)
20 mg/ml proteinase K
Buffered phenol (unit 2.1)
1:1 phenol/chloroform
3 M sodium acetate, pH 6.0 (appendix 2)
95% and 70% ethanol, ice-cold
TE buffer, pH 7.8 (appendix 2)
HindIII restriction endonuclease (unit 3.1) and appropriate buffer
12-well tissue culture plates
Additional reagents and equipment for preparing BS-C-1 cells for vaccinia infection (see Basic Protocol 1), tissue culture and counting cells (appendix 3F), phenol/chloroform extraction of DNA (unit 2.1), restriction endonuclease digestion of DNA (unit 3.1), Southern blotting (unit 2.9A), and hybridization (unit 2.10)

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 3: Detection of Vaccinia DNA Using Dot-Blot Hybridization

Materials

0.5 N NaOH
1 M Tris×Cl, pH 7.5 (appendix 2)
2× SSC (appendix 2)
Nitrocellulose membrane (see Table 2.9.1)
Whatman 3MM filter paper
80°C vacuum oven

Additional reagents and equipment for preparing cells, infecting with vaccinia virus, and performing selection (see Basic Protocol 1), dot blotting (unit 2.9B), and hybridization (unit 2.10)

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Alternate Protocol: Detection of Expressed Protein by a Dot-Blot Procedure

Additional Materials (also see Basic Protocol 3)

PBS/Tween (see recipe) with and without 4% (w/v) BSA
Antibody to foreign protein
100 µCi/ml [¹²⁵I]-labeled protein A (30 mCi/mg; Amersham)

Additional reagents and equipment for autoradiography (appendix 3A)

Basic Protocol 4: Detection of Expressed Protein Using Immunoblotting

Materials

Confluent BS-C-1 cell monolayer (unit 16.16)
Complete MEM-5 medium (unit 16.16)
Recombinant vaccinia virus stock (unit 16.16)
0.25 mg/ml trypsin (2× crystallized and salt-free;Worthington; filter sterilize and store at –20°C)
Cell lysis buffer (see recipe)
5× SDS sample buffer (unit 10.2)

6-well tissue culture plates
Sorvall centrifuge with H-6000A rotor (or equivalent)
95°C water bath

Additional reagents and equipment for preparing cell cultures for infection (see Basic Protocol 1) and immunoblotting (unit 10.8)

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 5: Detection of Expressed Protein Using Immunoprecipitation

Materials

Complete MEM-5 medium (unit 16.16)
Complete methionine- or cysteine-free MEM-5 medium (see recipe in unit 16.16 but use methionine- or cysteine-free MEM, e.g., from Life Technologies) prepared with dialyzed FBS (e.g., HyClone)
[³⁵S]methionine or [³⁵S]cysteine (depending on which amino acid was omitted from the medium; see Critical Parameters)
Phosphate-buffered saline (PBS; appendix 2)
Cell lysis buffer (see recipe)
Additional reagents and equipment for preparing cells and infecting with vaccinia virus (see Basic Protocol 4) and immunoprecipitation (unit 10.16)

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

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Literature Cited

Literature Cited
	Barrett, N., Mitterer, A., Mundt, W., Eibl, J., Eibl, M., Gallo, R.C., Moss, B., and Dorner, F. 1989. Large-scale production and purification of a vaccinia recombinant-derived HIV-1 gp 160 and analysis of its immunogenicity. AIDS Res. Hum. Retroviruses 5:159-171.
	Zhang, Y. and Moss, B. 1991. Inducer-dependent conditional-lethal mutant animal viruses. Proc. Natl. Acad. Sci. U.S.A. 88:1511-1515.