Construction of Deletion-knockout Mutant Fowlpox Virus (FWPV)
缺失剔除突变禽痘病毒(FWPV)的建立
发布: 2014年05月20日第4卷第10期 DOI: 10.21769/BioProtoc.1126 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:
May 2013
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Abstract
The construction of deletion-knockout poxviruses is a useful approach to determining the function of specific virus genes. This protocol is an adaptation of the transient dominant knockout selection protocol published by Falkner and Moss (1990) for use with vaccinia virus. The protocol makes use of the dominant selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) gene (Mulligan and Berg, 1981), under the control of an early/late poxvirus promoter. The deletion viruses that are produced no longer contain a selectable marker, which may be preferable for the production of vaccines.
Keywords: Knockout (基因敲除)
Fowlpox (鸡痘)
Deletion (删除)
Poxvirus (痘病毒)
Transient dominant (短暂的优势)
Materials and Reagents
- Primary chick embryo fibroblast cells (CEFs)
Notes:- Prepared from specific pathogen free quality embryos (10 days old)
- For a protocol to prepare CEFs see http://cshprotocols.cshlp.org/content/2006/2/pdb.prot4475.full
- Prepared from specific pathogen free quality embryos (10 days old)
- Fowlpox virus (FWPV) (e.g. FP9 strain) (GenBank accession number: AJ581527 )
- Gene specific oligonucleotides
- High fidelity taq polymerase (e.g. Q5, New England Biolabs, catalog number: M0491S )
- Qiaquick PCR purification kit (QIAGEN, catalog number: 28104 )
- T4 DNA ligase (New England Biolabs, catalog number: M0202T )
- Restriction enzymes (New England Biolabs)
- 199 media (Life Technologies, catalog number: 31150-022 )
- DMEM (Life Technologies, catalog number: 11995-065 )
- 10% tryptose phosphate broth (Sigma-Aldrich, catalog number: T8159 )
- Penicillin/Streptomycin (Life Technologies, catalog number: 15140122 )
- Nystatin (Sigma-Aldrich, catalog number: N1638 )
- NewBorn bovine serum (Life Technologies, catalog number: 16010-167 )
- Poxvirus recombination vector e.g. pGNR (as described in Reference 1)
- Transfection reagent e.g. Polyfect (QIAGEN, catalog number: 301105 )
- Mycophenolic acid (Sigma-Aldrich, catalog number: M5255 )
- Xanthine (Sigma-Aldrich, catalog number: X3627 )
- Hypoxanthine (Sigma-Aldrich, catalog number: H9636 )
- Low melting point agarose (Sigma-Aldrich, catalog number: A4018 )
- 10x MEM (Life Technologies, catalog number: 21430-020 )
- L-glutamine (Life Technologies, catalog number: 25030-024 )
- Sodium bicarbonate (Life Technologies, InvitrogenTM, catalog number: 25080-060 )
- Wizard® SV genomic DNA purification kit (Promega Corporation, catalog number: A2361 )
- Taq DNA polymerase (Life Technologies, InvitrogenTM, catalog number: 10342053 )
- MXH media (see Recipes)
- Overlay medium (see Recipes)
- Complete 199 media (see Recipes)
Equipment
- Thermal Cycler
- Microcentrifuge
- T25 cell culture flask
- -80 °C freezer
- Cell culture 6 well plates
- Marker pen
- 37 °C 5% CO2 cell culture incubator
- Sterile screw cap microfuge tubes
- Pipette tips (ART® 200 G)
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Laidlaw, S. M. and Skinner, M. A. (2014). Construction of Deletion-knockout Mutant Fowlpox Virus (FWPV). Bio-protocol 4(10): e1126. DOI: 10.21769/BioProtoc.1126.
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分类
微生物学 > 微生物遗传学 > 诱/突变
分子生物学 > DNA > 诱/突变
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