FWPV 嵌合 MVA的建立

Karen R. Buttigieg; Michael  A. Skinner

doi:10.21769/BioProtoc.1376

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Peer-reviewed

Construction of FWPV Chimaeric MVA

FWPV 嵌合 MVA的建立

KB Karen R. Buttigieg email

Michael A. Skinner

发布: 2015年01月05日第5卷第1期 DOI: 10.21769/BioProtoc.1376 浏览次数: 9066

评审: Anonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of Journal of Virology, featuring study using the protocol.

May 2013

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Abstract

Construction of chimaeric MVA is a useful tool with which to study gene function of related viruses. The protocol given here describes MVA chimaeras containing genes from Fowlpox virus (FWPV), although this can be applied to DNA derived from other organisms. There are a number of steps required to make the chimaeric MVA: 1) Purification of viral particles; 2) Extraction of DNA from purified viral particles; 3) Assembly of linear recombination templates; 4) Transfection of linear recombination templates; 5) Selection of chimaeric MVA.
Note: This procedure uses live virus, and should be conducted using Good Microbiological Practice, in accordance with international and national biocontainment requirements. This procedure also involves Genetic Modification of microorganisms, and appropriate safety approval should be obtained before commencing.

Materials and Reagents

Purification of viral particles
1. Fowlpox virus (FWPV) (e.g. ATCC, catalog number: VR-251 )
2. Modified Vaccinia virus Ankara (MVA) (ATCC, catalog number: VR-1508 )
3. CEF cells (Institute for Animal Health)
4. Tris-EDTA buffer (Sigma-Aldrich, catalog number: 93283 )
5. DMEM (e.g. Sigma-Aldrich, catalog number: D6429 )
6. Foetal bovine serum (FBS) (e.g. Sigma-Aldrich, catalog number: F9665 )
7. 10x medium 199 with Earle’s salts (Sigma-Aldrich, catalog number: M0650 )
8. Heat Inactivated newborn bovine serum (Life Technologies, catalog number: 26010074 )
9. Tryptose phosphate broth (Sigma-Aldrich, catalog number: T8159 )
10. Nystatin (Sigma-Aldrich, catalog number: N1638 )
11. Penicillin-streptomycin solution (Sigma-Aldrich, catalog number: P4333 )
12. L-glutamine solution (Sigma-Aldrich, catalog number: G7513 )
13. Sodium bicarbonate (Sigma-Aldrich, catalog number: S8761 )
14. Amicon Ultra-15 Centrifugal filter unit (100 kDa membrane NMWL) (Merck Millipore, catalog number: UFC910024 )
15. FWPV virus growth medium (see Recipes)
16. MVA virus growth medium (see Recipes)
17. TE (see Recipes)
Extraction of DNA from purified viral particles
1. Phenol saturated with 10 mM Tris-HCl (Sigma-Aldrich, catalog number: P4557 )
2. Phenol-chloroform-isoamyl alcohol (25:24:1, saturated with 10 mM Tris, pH 8.0) (Sigma-Aldrich, catalog number: P2069 )
3. Chloroform-isoamyl alcohol (24:1) (Sigma-Aldrich, catalog number: 25666 )
4. 3 M Sodium acetate (Sigma-Aldrich, catalog number: 71196 )
5. Ethanol (Sigma-Aldrich, catalog number: E7023 )
6. 10 mM Tris-Cl (pH 8.5) (QIAGEN, catalog number: 19086 )
7. Tris-HCl (Sigma-Aldrich, catalog number: T5941 )
8. EDTA (Sigma-Aldrich, catalog number: EDS )
9. NaCl (Sigma-Aldrich, catalog number: S3014 )
10. SDS solution (Sigma-Aldrich, catalog number: 71736 )
11. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
12. Proteinase K (Sigma-Aldrich, catalog number: P2308 )
13. 2x extraction buffer (see Recipes)
Assembly of linear recombination templates
1. PCR oligonucleotides (Sigma-Aldrich)
2. Accuprime Taq high fidelity polymerase (Life Technologies, catalog number: 12346086 )
3. pNEB193 (New England Biolabs)
4. Plasmid encoding Escherichia coli (E.coli) gpt under p7.5 promoter (e.g. pGNR, available on request)
5. QIAquick PCR purification kit (QIAGEN, catalog number: 28104 )
6. QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
Transfection of linear recombination templates
1. DF-1 cells (ATCC, catalog number: CRL-12203 )
2. DMEM (e.g. Sigma-Aldrich, catalog number: D6429)
3. Foetal bovine serum (FBS) (e.g. Sigma-Aldrich, catalog number: F9665)
4. Penicillin-streptomycin solution (Sigma-Aldrich, catalog number: P4333)
5. Salmon sperm DNA solution (Life Technologies, catalog number: 15632011 )
6. Opti-MEM (Life Technologies, catalog number: 31985062 )
7. Polyfect (QIAGEN, catalog number: 301105 )
8. DF-1 culture medium (see Recipes)
Selection of chimaeric MVA
1. DMEM (e.g. Sigma-Aldrich, catalog number: D6429)
2. Foetal bovine serum (FBS) (e.g. Sigma-Aldrich, catalog number: F9665)
3. Penicillin-streptomycin solution (Sigma-Aldrich, catalog number: P4333)
4. Mycophenolic acid (Sigma-Aldrich, catalog number: M5255 )
5. Xanthine (Sigma-Aldrich, catalog number: X3627 )
6. Hypoxanthine (Sigma-Aldrich, catalog number: H9636 )
7. Hydrochloric acid (Sigma-Aldrich, catalog number: H9892 )
8. Sodium hydroxide (Sigma-Aldrich, catalog number: S2770 )
9. 2% DMEM + MXH (see Recipes)

Equipment

Benchtop centrifuge (e.g. Hettich^®, model: Rotanta 460 )
Thermal cycler (Techne Progene^®)
Laminar flow microbiological safety cabinet (e.g. HeraSafe^®, model: 51022738 )
Humidified CO₂ incubator (e.g. Thermo Fisher Scientific, model: BB15 )
Cuphorn sonicator (e.g. Ultrasonics^TM, model: W-375 )