Construction of Deletion-knockout Mutant Fowlpox Virus (FWPV)

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Journal of Virology
May 2013



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

  1. Primary chick embryo fibroblast cells (CEFs)
    1. Prepared from specific pathogen free quality embryos (10 days old)
    2. For a protocol to prepare CEFs see
  2. Fowlpox virus (FWPV) (e.g. FP9 strain) (GenBank accession number: AJ581527 )
  3. Gene specific oligonucleotides
  4. High fidelity taq polymerase (e.g. Q5, New England Biolabs, catalog number: M0491S )
  5. Qiaquick PCR purification kit (QIAGEN, catalog number: 28104 )
  6. T4 DNA ligase (New England Biolabs, catalog number: M0202T )
  7. Restriction enzymes (New England Biolabs)
  8. 199 media (Life Technologies, catalog number: 31150-022 )
  9. DMEM (Life Technologies, catalog number: 11995-065 )
  10. 10% tryptose phosphate broth (Sigma-Aldrich, catalog number: T8159 )
  11. Penicillin/Streptomycin (Life Technologies, catalog number: 15140122 )
  12. Nystatin (Sigma-Aldrich, catalog number: N1638 )
  13. NewBorn bovine serum (Life Technologies, catalog number: 16010-167 )
  14. Poxvirus recombination vector e.g. pGNR (as described in Reference 1)
  15. Transfection reagent e.g. Polyfect (QIAGEN, catalog number: 301105 )
  16. Mycophenolic acid (Sigma-Aldrich, catalog number: M5255 )
  17. Xanthine (Sigma-Aldrich, catalog number: X3627 )
  18. Hypoxanthine (Sigma-Aldrich, catalog number: H9636 )
  19. Low melting point agarose (Sigma-Aldrich, catalog number: A4018 )
  20. 10x MEM (Life Technologies, catalog number: 21430-020 )
  21. L-glutamine (Life Technologies, catalog number: 25030-024 )
  22. Sodium bicarbonate (Life Technologies, InvitrogenTM, catalog number: 25080-060 )
  23. Wizard® SV genomic DNA purification kit (Promega Corporation, catalog number: A2361 )
  24. Taq DNA polymerase (Life Technologies, InvitrogenTM, catalog number: 10342053 )
  25. MXH media (see Recipes)
  26. Overlay medium (see Recipes)
  27. Complete 199 media (see Recipes)


  1. Thermal Cycler
  2. Microcentrifuge
  3. T25 cell culture flask
  4. -80 °C freezer
  5. Cell culture 6 well plates
  6. Marker pen
  7. 37 °C 5% CO2 cell culture incubator
  8. Sterile screw cap microfuge tubes
  9. Pipette tips (ART® 200 G)


  1. Construction of recombination plasmid encoding deletion of the gene of interest (GoI)
    1. Design four oligonucleotide primers
      1. Primer 1: Forward primer to bind 500 bp upstream of GoI and including 5’ recombination vector compatible restriction site.
      2. Primer 2: Reverse primer to bind 20 bp downstream of GoI start codon and including a 5’ tail complementary to primer 3.
      3. Primer 3: Forward primer to bind 30-50 bp upstream of GoI stop codon and designed to disrupt the open reading frame (ORF), by introduction of a frameshift, and including a 5’ tail complementary to primer 2.
      4. Primer 4: Reverse primer to bind 500 bp downstream of GoI and including a 5’ recombination vector compatible restriction site.
        Note: Care should be taken to ensure primers 2 and 3 do not disrupt any putative early/late transcription initiation or termination sites.
    2. Using PCR and a high fidelity polymerase, separately amplify PCR products (using manufacturers' recommendations) from purified FWPV template DNA using primer pairs 1 & 2 and 3 & 4.
    3. Purify the PCR products from step A2 using Qiaquick PCR purification (according to the manufacturers' recommendations).
    4. Combine equimolar amounts of the PCR products from step A3 and carry out a further PCR using primers 1 & 4. This will result in a single PCR product of approximately 1.1 kb consisting of a region of the FWPV genome deleted for the GoI.
    5. Purify the PCR product from step A4 using Qiaquick PCR purification.
    6. Ligate the PCR product into the recombination vector (pGNR) using the selected/designed restriction enzymes (for guidance see Reference 4).

  2. Making the recombinant fowlpox virus
    Due to matching sequences between the recombination vector and fowlpox virus genome homologous recombination occurs resulting in recombinant viruses (A scheme of the recombination events can be found in Figure 4, Reference 2.).
    1. Seed a T25 tissue culture flask with 3.9 x 106 CEFs (to achieve 70% confluency the following day) in 5 ml 10% serum 199 media.
    2. Remove media and infect CEFs with FWPV (to give a multiplicity of infection of 3 to 5) in 0.5 ml of serum-free DMEM.
    3. Incubate for 2 h at 37 °C, 5% CO2 with occasional rocking.
    4. Add 3 ml 2% serum 199 media and leave for a further 2 h.
    5. Prepare transfection mix [DNA (from step A6): 2 µg; serum-free DMEM: 200 µl; Polyfect: 10 µl], pipette up and down a few times and leave at room temperature for 15-20 min (
    6. Remove media from cells and wash with serum free DMEM media.
    7. Add 3 ml 2% serum 199 media to cells.
    8. Add transfection mix directly to flask and rock a few times to mix.
    9. Incubate 37 °C, 5% CO2 overnight.
    10. The following day, replace the media in the T25 with 5 ml 2% serum 199 MXH media and incubate 37 °C, 5% CO2 for a further 3 days.
    11. Release progeny virus from cells by freeze thawing three times using a -80 °C freezer. The protocol can be stopped at this stage and resumed when purification of the virus is required. It is not necessary to centrifuge the freeze-thawed mixture to remove cell debris.

  3. Purification of recombinant virus
    1. Seed a 6 well plate with 1.2 x 107 CEFs (2 x 106 cells in 2 ml/well) (to achieve 100% confluency the following day) in 10% 199 media.
    2. Prepare 10 fold serial dilutions in serum-free DMEM (10-1 to 10-6) of the freeze-thawed transfected/infected cell supernatant containing progeny virus.
    3. Remove media from the cells and add 1 ml of the progeny virus dilutions to the wells of the 6 well plate.
    4. Incubate for 2 h at 37 °C, 5% CO2 with occasional rocking. Remove virus and overlay with 1% agarose overlay media containing MXH. For the preparation of this medium incubate 2x MEM (containing the MXH solutions) at 37 ºC and 2% low gelling temperature agarose at 42 °C. Upon removal of virus from infected CEFs combine the 2x MEM (containing the MXH solutions) and 2% agarose solution in the tissue culture cabinet and overlay cells (3 ml per well of a 6 well plate). Allow agarose to set before incubating at 37 °C, 5% CO2 until plaques are visible (5 to 6 days).
      Note: Importantly and unusually, FWPV plaques are visible as opaque, not clear, areas! Their presence can be confirmed by broad field microscopy, preferably under phase contrast. Note also that MXH causes FWPV to replicate more slowly, resulting in smaller plaques so do not just select large plaques.
    5. By eye mark the location of isolated plaques on the bottom of the plates with a fine marker pen, then pick individual plaques using cut-off filter tips (e.g. ART 200 G max, volume 200 µl) into 500 µl serum free DMEM.
    6. Freeze thaw the isolated plaque suspension three times (as previously).
    7. Repeat steps C2-6 three times in order to ensure the virus is sufficiently homogenous.
      Note: The above steps isolate single-crossover intermediate recombinant FWPV, each carrying two copies of the GoI, one of which is mutant, the other parental (A scheme of the recombination events can be found in Figure 4 in Reference 2.). In order to resolve the intermediate virus to derivatives lacking gpt and containing either parental or mutant GoI, further plaque purification in the absence of MXH selection is needed. Assuming the GoI is not essential or required for FWPV replication and plaque production in CEF, resolved derivatives containing parental or mutant GoI will be isolated at equal frequencies, as long as primers 1 and 4 are placed equidistant from the GoI. Equivalence of isolation frequency is therefore conversely a test of whether a GoI plays a significant role in FWPV replication and plaque production in CEF.
    8. Repeat plaque purification three further times in the absence of MXH.
    9. Test plaques to determine if intermediate recombinant FWPV have resolved by infecting CEFs in 6 well plates with 100 µl freeze thawed plaque purified virus (diluted in 2 ml 2% serum 199 media) and incubating 37 °C, 5% CO2 for 4 days.
    10. Remove media from infected CEF and wash cells with PBS.
    11. Using a Wizard® SV genomic DNA purification kit lyse cells using 300 µl of supplied lysis buffer and isolate FWPV DNA (as per manufacturers' instructions -
    12. Using 2 µl of extracted genomic DNA carry out PCR using the original flanking primers (1 & 4) and separate by agarose electrophoresis. The resultant amplicons will represent either parental (GoI + 1,100 bp) or mutant (in this case deleted) genotype (1,100 bp) for the GoI. To ensure that the recombinant is homogeneous and lacking the gpt gene, a further PCR can be carried out using a flanking primer (1 or 4) in combination with an appropriate gpt internal primer. This PCR assay should result in no amplicon.
      Note: It may be necessary to carry out further plaque purifications in the absence of MXH until the virus resolves. If it is suspected that the GoI might be essential it would be prudent to perform parallel plaque purifications from 10 to 20 intermediate recombinant plaques. Should PCR screening indicate that all of the derivatives contain parental GoI, then it is probable that the GoI is essential to FWPV replication and plaque production in CEF.


  1. MXH media
    Stock solutions:
    Mycophenolic acid 10 mg/ml in 0.1 M NaOH
    Xanthine 10 mg/ml in 0.1 M NaOH
    Hypoxanthine 6 mg/ml in 0.1 M NaOH
    Filter sterilise all solutions
    To 100 ml (final) of medium:
    0.25 ml Mycophenolic acid (final conc: 0.025 mg/ml)
    2.5 ml Xanthine (final conc: 0.25 mg/ml)
    0.25 ml Hypoxanthine (final conc: 0.015 mg/ml)
    Note: Mycophenolic acid is toxic, therefore wear gloves and rinse glassware thoroughly after use.
  2. Overlay medium
    Note: Overlay medium is prepared by the mixing of 2x MEM and 2% low gelling temperature agarose. For media that requires the addition of MXH, this is added to the 2x MEM, before mixing with the agarose.
    2x MEM (50 ml):
    10 ml 10x MEM
    2.3 ml 7.5% sodium bicarbonate
    1 ml L-Glutamine
    0.1 ml Penicillin/Streptomycin
    2.5 ml Nystatin
    2 ml newborn bovine serum
    32.1 ml water
    Mix and then hold medium at 37 °C
    2% low gelling temperature agarose:
    1 g agarose in 50 ml distilled water, autoclaved and held at 42 °C
    Note: Stocks of 2% agarose may be prepared in advance and melted just prior to use in a boiling water bath.
  3. Complete 199 media
    1x 199 media:
    10% tryptose phosphate broth
    1% Penicillin-Streptomycin
    2.5% Nystatin
    10% (for cell growth) or 2% (for cell maintenance) newborn bovine serum


This protocol was adapted from Boulanger et al. (1998); Falkner and Moss (1990); and Laidlaw et al. (2013). This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC), grants BBS/B/00115/2, BB/E009956/1, and BB/G018545/1.


  1. Boulanger, D., Green, P., Smith, T., Czerny, C. P. and Skinner, M. A. (1998). The 131-amino-acid repeat region of the essential 39-kilodalton core protein of fowlpox virus FP9, equivalent to vaccinia virus A4L protein, is nonessential and highly immunogenic. J Virol 72(1): 170-179.
  2. Falkner, F. G. and Moss, B. (1990). Transient dominant selection of recombinant vaccinia viruses. J Virol 64(6): 3108-3111.
  3. Fowlpox virus FP9, GenBank accession number: AJ581527.
  4. He, F. L. (2011). Standard DNA cloning. Bio-protocol 1(7): e52.
  5. Laidlaw, S. M., Robey, R., Davies, M., Giotis, E. S., Ross, C., Buttigieg, K., Goodbourn, S. and Skinner, M. A. (2013). Genetic screen of a mutant poxvirus library identifies an ankyrin repeat protein involved in blocking induction of avian type I interferon. J Virol 87(9): 5041-5052.
  6. Mulligan, R. and Berg, P. (1981). Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A 78(4): 2072-2076.


缺失敲除痘病毒的构建是确定特定病毒基因的功能的有用方法。 该方案是由Falkner和Moss(1990)公布的用于牛痘病毒的瞬时显性敲除选择方案的改编。 该方案利用在早期/晚期痘病毒启动子控制下的显性选择标记大肠杆菌鸟嘌呤磷酸核糖基转移酶(gpt)基因(Mulligan和Berg,1981)。 产生的缺失病毒不再含有可选择标记,其可优选用于疫苗的生产。

关键字:基因敲除, 鸡痘, 删除, 痘病毒, 短暂的优势


  1. 原代鸡胚成纤维细胞(CEF)
    1. 从特定病原体免费质量胚胎(10天龄)制备
    2. 有关准备CEF的协议,请参阅 http://cshprotocols.cshlp .org/content/2006/2/pdb.prot4475.full
  2. 禽痘病毒(FWPV)(例如FP9株)(GenBank登录号:AJ581527)
  3. 基因特异性寡核苷酸
  4. 高保真taq聚合酶(例如,Q5,New England Biolabs,目录号:M0491S)
  5. Qiaquick PCR纯化试剂盒(QIAGEN,目录号:28104)
  6. T4 DNA连接酶(New England Biolabs,目录号:M0202T)
  7. 限制酶(New England Biolabs)
  8. 199培养基(Life Technologies,目录号:31150-022)
  9. DMEM(Life Technologies,目录号:11995-065)
  10. 10%磷酸胰蛋白酶肉汤(Sigma-Aldrich,目录号:T8159)
  11. 青霉素/链霉素(Life Technologies,目录号:15140122)
  12. 制霉菌素(Sigma-Aldrich,目录号:N1638)
  13. NewBorn牛血清(Life Technologies,目录号:16010-167)
  14. 痘病毒重组载体例如pGNR(如参考文献1中所述)
  15. 转染试剂(例如) Polyfect(QIAGEN,目录号:301105)
  16. 霉酚酸(Sigma-Aldrich,目录号:M5255)
  17. 黄嘌呤(Sigma-Aldrich,目录号:X3627)
  18. 次黄嘌呤(Sigma-Aldrich,目录号:H9636)
  19. 低熔点琼脂糖(Sigma-Aldrich,目录号:A4018)
  20. 10×MEM(Life Technologies,目录号:21430-020)
  21. L-谷氨酰胺(Life Technologies,目录号:25030-024)
  22. 碳酸氢钠(Life Technologies,Invitrogen TM,目录号:25080-060)
  23. Wizard SV基因组DNA纯化试剂盒(Promega Corporation,目录号:A2361)
  24. Taq DNA聚合酶(Life Technologies,Invitrogen TM,目录号:10342053)
  25. MXH媒体(见配方)
  26. 覆盖介质(参见配方)
  27. 完成199媒体(参见配方)


  1. 热循环仪
  2. 微量离心机
  3. T25细胞培养瓶中
  4. -80°C冰箱
  5. 细胞培养6孔板
  6. 记号笔
  7. 37℃5%CO 2细胞培养孵育器
  8. 无菌螺旋盖微量离心管
  9. 移液器提示(ART ® 200 G)


  1. 构建编码目的基因缺失的重组质粒(GoI)
    1. 设计四个寡核苷酸引物
      1. 引物1:正向引物结合Go1上游500bp,包括5'重组载体相容限制性位点
      2. 引物2:反向引物结合Go1起始密码子下游20bp并包括与引物3互补的5'尾。
      3. 引物3:正向引物结合GoI终止密码子上游30-50bp并设计为通过引入移码并破坏开放阅读框(ORF),并包括与引物2互补的5'尾。
      4. 引物4:反向引物以结合Go1下游500bp并包括5'重组载体相容性限制性位点 注意:应注意确保引物2和3不会破坏任何推定的早期/晚期转录起始或终止位点。
    2. 使用PCR和高保真聚合酶,使用引物对1和2扩增来自纯化的FWPV模板DNA的PCR产物(使用制造商的建议) 2和3& 4.
    3. 使用Qiaquick PCR纯化(根据制造商的建议)纯化步骤A2的PCR产物
    4. 合并等摩尔量的来自步骤A3的PCR产物,并使用引物1和2进行进一步的PCR。 这将导致大约1.1kb的单一PCR产物,其由为GoI删除的FWPV基因组的区域组成。
    5. 使用Qiaquick PCR纯化纯化来自步骤A4的PCR产物
    6. 使用选择/设计的限制性内切酶将PCR产物引入重组载体(pGNR)(参考文献4)。

  2. 制造重组禽痘病毒
    1. 在5ml 10%血清199培养基中接种具有3.9×10 6个CEF的T25组织培养瓶(第二天达到70%融合)。
    2. 取出培养基,并用0.5ml无血清DMEM中的FWPV感染CEF(使感染复数达到3至5)。
    3. 在37℃,5%CO 2下孵育2小时,偶尔摇摆。
    4. 加入3ml 2%血清199培养基,并再静置2小时
    5. 准备转染混合物[DNA(来自步骤A6):2μg; 无血清DMEM:200μl; Polyfect:10μl],向上和向下移液几次,并在室温下放置15-20分钟( .aspx?id = bf924409-51f9-4bd0-b63f-a8369c70a331& lang = en )。
    6. 从细胞中取出培养基,用无血清DMEM培养基洗涤
    7. 向细胞中加入3ml 2%血清199培养基
    8. 将转染混合物直接加入烧瓶中并摇动几次混合
    9. 在37℃,5%CO 2保温过夜。
    10. 第二天,用5ml 2%血清199MXH培养基替换T25中的培养基,并在37℃,5%CO 2下再培养3天。
    11. 通过使用-80℃冷冻器冷冻解冻三次从细胞释放后代病毒。 该方案可以在该阶段停止,并且当需要纯化病毒时恢复。 不必离心冷冻融化的混合物以除去细胞碎片。

  3. 重组病毒的纯化
    1. 将具有1.2×10 7个CEF(2×10 6个细胞,2ml /孔)(在第二天达到100%融合)的6孔板在10% 199媒体
    2. 在含有后代病毒的冻融转染/感染的细胞上清液的无血清DMEM(10μl〜10μl,10μl〜6μl)中制备10倍系列稀释液。
    3. 从细胞中除去培养基,并将1ml子代病毒稀释液加入6孔板的孔中
    4. 在37℃,5%CO 2下孵育2小时,偶尔摇动。用含有MXH的1%琼脂糖覆盖培养基除去病毒和覆盖物。对于该培养基的制备,在37℃和2%低胶凝温度琼脂糖上在42℃温育2×MEM(含有MXH溶液)。从感染的CEF中除去病毒后,组织培养箱中的2×MEM(含有MXH溶液)和2%琼脂糖溶液和覆盖细胞(6孔板的每孔3ml)。使琼脂糖凝固,然后在37℃,5%CO 2孵育,直到可见斑块(5至6天)。
    5. 通过用细标记笔眼睛标记板底部上的孤立噬菌斑的位置,然后使用截止过滤器尖端(例如ART 200G max,体积200μl)将单个噬菌斑挑选到500 μl无血清DMEM
    6. 将分离的菌斑悬浮液冻融三次(如前所述)。
    7. 重复步骤C2-6三次,以确保病毒足够均匀。
      注意:上述步骤分离单交换中间体重组FWPV,每个携带两个拷贝的GoI,其中一个是突变体,另一个是亲本(重组事件的方案可参见参考文献2中的图4。 )。为了将中间体病毒解析为缺少gpt并且含有亲本或突变体Go1的衍生物,需要在不存在MXH选择的情况下进一步的噬斑纯化。假设GoI不是必需的或必需的 对于CEF中的FWPV复制和斑块产生,包含亲本或突变体GoI的解析衍生物将以相等的频率分离,只要引物1和4与GoI等距离。因此,相反地,隔离频率的等效性是测试GoI在CEF中FWPV复制和斑块产生中是否起重要作用。
    8. 在没有MXH的情况下重复进行三次空斑纯化。
    9. 通过用100μl冷冻融化的噬菌斑纯化的病毒(稀释在2ml 2%血清199培养基中)并在37℃,5%CO 2中孵育来检测6孔板中的CEF是否已经解析中间体重组FWPV的测试噬斑。/sub> 4天。
    10. 从感染的CEF中删除媒体,用PBS洗涤细胞
    11. 使用Wizard SV基因组DNA纯化试剂盒,使用300μl提供的裂解缓冲液裂解细胞,并分离FWPV DNA(根据制造商的说明 -。
    12. 使用2μl提取的基因组DNA使用原始侧翼引物(1& 4)进行PCR,并通过琼脂糖电泳分离。所得扩增子将代表GoI的亲代(GoI + 1,100bp)或突变(在这种情况下缺失)基因型(1,100bp)。为了确保重组体是均质的并且缺乏gpt基因,可以使用侧翼引物(1或4)与合适的gpt内部引物组合进行进一步的PCR。该PCR测定应该不产生扩增子 注意:可能有必要在没有MXH的情况下进行进一步的斑块纯化,直到病毒消退。如果怀疑GoI可能是必需的,则可以谨慎地进行10至20个中间重组噬斑的平行噬菌斑纯化。如果PCR筛选表明所有衍生物含有亲本GoI,那么可能GoI对于CEF中的FWPV复制和斑块产生是必需的。


  1. MXH媒体
    霉酚酸10mg/ml在0.1M NaOH中
    黄嘌呤10mg/ml在0.1M NaOH中
    次黄嘌呤6mg/ml在0.1M NaOH中
    0.25ml霉酚酸(最终浓度:0.025mg/ml) 2.5ml黄嘌呤(最终浓度:0.25mg/ml) 0.25ml次黄嘌呤(最终浓度:0.015mg/ml) 注意:霉酚酸有毒,因此戴上手套,并在使用后彻底冲洗玻璃器皿。
  2. 覆盖媒介
    注:通过混合2x MEM和2%低胶凝温度琼脂糖制备覆盖培养基。 对于需要添加MXH的培养基,将其加入到2x MEM中,然后与琼脂糖混合。
    10ml 10×MEM
    2.3ml 7.5%碳酸氢钠 1 ml L-谷氨酰胺 0.1ml青霉素/链霉素 2.5ml制霉菌素
    2 ml新生牛血清
    混合,然后在37℃保持培养基 2%低凝胶化温度琼脂糖:
    1g琼脂在50ml蒸馏水中,高压灭菌并保持在42℃ 注意:2%琼脂糖储备可以提前制备并在即将用于沸水浴之前熔化。
  3. 完成199媒体
    1x 199媒体:
    1%青霉素 - 链霉素 2.5%制霉菌素


该方案改编自Boulanger等人(1998); Falkner和Moss(1990); 和Laidlaw等人(2013)。 这项工作由生物技术和生物资助 科学研究委员会(BBSRC),授予BBS/B/00115/2,BB/E009956/1和BB/G018545/1。


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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.