FWPV 嵌合 MVA的建立

Karen R. Buttigieg; Michael  A. Skinner

doi:10.21769/BioProtoc.1376

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
登录
/
注册
- 个人主页
- 编辑个人信息
- 修改密码
- 退出
CN
- EN - English
- CN - 中文

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: 9518

评审: Anonymous reviewer(s)

PDF

Q&A

引用

Cited by

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

Submit your protocol to the Special Issue on Technologies for RNA Detection

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

全血HIV-2 DNA的定量分析

Zsófia Szojka [...] Patrik Medstrand

2019年10月20日 6004 阅读

使用 CRISPR/Cas9 筛选牛痘病毒重组体

Anjali Gowripalan [...] David C. Tscharke

2021年12月20日 3417 阅读

用甘蔗花叶病毒构建体接种玉米并应用于秋季棉铃虫的RNA干扰

Iram Gull and Georg Jander

2023年07月20日 2206 阅读

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 )