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Detection of piggyBac-mediated Transposition by Splinkerette PCR in Transgenic Lines of Strongyloides ratti
采用Splinkerette PCR检测类圆线虫转基因株系中的piggyBac介导转位   

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Abstract

Splinkerette PCR (spPCR) is a newly developed and efficient method to ascertain and characterize genomic insertion sites of transgenes. The method described in this protocol was successfully applied to confirm piggyBac transposon-mediated integration of transgenes into chromosomes of the parasitic nematode Strongyloides ratti. This work is described in detail in Shao et al. (2012) and presented here in a simplified diagram (Figure 1). Using this method, chromosomal loci of integration were determined based on target site and 5’- and 3’ flanking sequences. Therefore, spPCR can be a useful method to confirm integrative transgenesis in functional genomic studies of parasitic nematodes. Potter and Luo (2010) contains a protocol for use of spPCR to detect and map piggyBac transposon-mediated chromosomal integrations in Drosophila, and was the source of our method for Strongyloides. The splinkerette- and piggyBac-specific oligos described in that reference could be used without modification in Strongyloides. For interested readers, a general review of the biology of parasitic nematodes in the genus Strongyloides may be found in Viney and Lok (2007), and a methods-based article on S. stercoralis as an experimental model, with information on transgenesis, may be found in Lok (2007).

Keywords: Strongyloides(粪), Transgenesis(转基因), Chromosomal integration(染色体整合), Transposon(转座子), Splinkerette PCR(splinkerette-PCR)



Figure 1. Diagrammatic representation of protocol for mapping transgene integrations in Strongyloides by splinkerette PCR (adapted from Potter and Luo, 2010)

Materials and Reagents

  1.  Free-living adult worms
  2. Genomic DNA extraction
    Gentra Puregene Tissue Kit (QIAGEN), including
    1. Cell lysis solution (catalog number: 8304295 )
    2. Protein precipitation solution (catalog number: 8273807 )
    3. DNA hydration solution (catalog number: 8274043 )
  3. Enzymes for digestion and treatment
    Restriction enzymes include BstY I, BamH I and Bgl II (New England Biolabs)
    Others include Shrimp Alkaline Phosphatase (United State Biological, catalog number: P4071-05 ) and Exonuclease I (New England Biolabs, catalog number: M0293S )
  4. Ligation reagents
    10x Ligase buffer and T4 Ligase (New England Biolabs, catalog number: M0202S )
  5. PCR reagents
    5x Phusion High-Fidelity Buffer, Phusion HF DNA Polymerase (New England Biolabs, catalog number: M0530S )
  6. Oligonucleotides and primers
    The oligonucleotides detailed in Table 1 must be synthesized   
    All sequences are from Potter and Luo (2010), Table S13
  7. TE buffer (New England Biolabs, catalog number: E6293 )
  8. 10x NEB Buffer 2
  9. 1% agarose gel

    Oligo or Primer  
    Sequence
    SPLNK-BOT  
     5’-CGAAGAGTAACCGTTGCTAGGAGAGACCGTGGCTGAATGAGACTGGTGTCGACACTAGTGG-3’
    SPLNK-GATC-TOP   
    5’-GATCCCACTAGTGTCGACACCAGTCTCTAATTTTTTTTTTCAAAAAAA-3’
    SPLNK#1  
    5’-CGAAGAGTAACCGTTGCTAGGAGAGACC-3’
    SPLNK#2   
    5’-GTGGCTGAATGAGACTGGTGTCGAC-3’
    3’SPLNK-PB#1   
    5’-GTTTGTTGAATTTATTATTAGTATGTAAG-3’
    5’SPLNK-PB#1   
    5’-ACCGCATTGACAAGCACG-3’
    3’SPLNK-PB#2   
    5’-CGATAAAACACATGCGTC-3’
    5’SPLNK-PB#2   
    5’-CTCCAAGCGGCGACTGAG-3’
    3’SPLNK-PB-SEQ   
    5’-ACGCATGATTATCTTTAAC-3’
    5’SPLNK-PB-SEQ   
    5’-CGACTGAGATGTCCTAAATGC-3’
    Table 1.  Oligonucleotides and primers for splinkerette PCR mapping of piggyBac transposon-mediated transgene integrations

Equipment

  1. PCR Thermal Cyclers (Mastercycler Personal, Eppendorf)
  2. Gel electrophoresis
  3. Gel documentation (FOTODYNE, model: FOTO/Analyst FX )

Procedure

  1. Collect stably transformed free-living adult Strongyloides from charcoal cultures of fresh host feces incubated at 22 °C for 48 h (Lok, 2007).  Extract genomic DNA (gDNA) from ~500 free-living adult worms using the Gentra Puregene Tissue Kit. Parasites do not need to be homogenized.  Isolated worms are pelleted at 14,000 x g and then substituted for the “ground tissue” in step 2, followed by step 2b of the manufacturer’s protocol accompanying the Gentra Puregene Tissue Kit, using reagent volumes recommended for tissue samples of 5-10 mg.  Resuspend extracted gDNA in 50 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to yield ~ 25 ng/μl.
  2. Digest Genomic DNA (~100 ng) with BstYI, BamHI, and Bgl II for 2 h in a total volume of 15 μl for each reaction (Figure 1A). Follow manufacturer’s instructions for reaction conditions and inactivation steps for each restriction enzyme.
  3. Add 5 μl of 100 μM SPLINK-BOT oligonucleotide, 5 μl 100 μM SPLINK-GATC-TOP oligonucleotide and 10x NEB Buffer 2 in a total reaction volume of 100 μl. Heat to 95 °C for 3 min then cool on bench to room temperature to anneal splinkerette oligonucleotides. Ligate digested gDNA with GATC sticky ends to annealed splinkerette oligonucleotides using T4 ligase for 8 h at 16 °C in a total volume of 30 μl (Figure 1B).
  4. Two rounds of spPCR are conducted following ligation of genomic DNA restriction fragments to splinkerette oligo nucleotides.
    1. For the first round of spPCR, combine primer SPLINK#1, which targets the ligated splinkerette oligonucleotides, with either primer 3’SPLNK-PB#1 or primer 5’SPLNK-PB#1. 3’SPLNK-PB#1 and 5’SPLNK-PB#1 target the 3’ and 5’ ends of the transgene sequences, respectively. Carry out the amplification with Phusion Polymerase and 5-20 ng of splinkerette-ligated genomic DNA as template. Thermal cycling conditions are:
      1. 98 °C for 1 min
      2. 98 °C for 20 sec
      3. 55 °C for 15 sec
      4. 72 °C for 2 min
      5. 29 more cycles from ii to iv
      6. a final extension at 72 °C for 10 min.
    2. The second round of spPCR is nested PCR carried out with primer SPLNK#2 paired with either primer 3’SPLNK-PB#2 or primer 5’SPLNK-PB#2 and a 1:20 dilution of spPCR products from the first-round spPCR as template. Carry out the second-round amplification with Phusion High-Fidelity DNA Polymerase using the same thermal cycling conditions as specified for first-round spPCR. Analyze all PCR products by 1% agarose gel electrophoresis.
    3. Incubate the products of second-round spPCR with 1 μl Shrimp Alkaline Phosphatase (1 U/ml) and 1 μl Exonuclease I (20,000 U/ml) at 37 °C for 2 h, and then at 80 °C for 15 min (Figure 1C).
    4. Sequence the treated PCR products directly using primers 3’SPLNK-PB-SEQ and 5’SPLNK-PB-SEQ, targeting the 3’ and 5’ ends of the transgene sequence, respectively. Identify chromosomal locations of integrations based on generated sequences and matching fragments in the genome database (Figure 1D, 1E).

Acknowledgments

This protocol details methods originally published in Shao et al. (2012). This work was funded by grant number AI050668 from the US National Institutes of Health.

References

  1. Lok, J. B. Strongyloides stercoralis: a model for translational research on parasitic nematode biology (February 17, 2007), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.134.1, http://www.wormbook.org.
  2. Potter, C. J. and Luo, L. (2010). Splinkerette PCR for mapping transposable elements in Drosophila. PLoS One 5(4): e10168. 
  3. Shao, H., Li, X., Nolan, T. J., Massey, H. C., Jr., Pearce, E. J. and Lok, J. B. (2012). Transposon-mediated chromosomal integration of transgenes in the parasitic nematode Strongyloides ratti and establishment of stable transgenic lines. PLoS Pathog 8(8): e1002871.
  4. Viney, M. E. and Lok, J. B. Strongyloides spp. (May 23, 2007), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.141.1, http://www.wormbook.org.

简介

Splinkerette PCR(spPCR)是一种新发展和有效的方法来确定和表征转基因的基因组插入位点。该方案中描述的方法被成功地应用于确认转座子介导的转基因整合到寄生线虫类线虫的染色体中的piggyBac 。该工作在Shao等人(2012)中详细描述并在简化图(图1)中呈现。使用该方法,基于靶位点和5'和3'侧翼序列确定整合的染色体基因座。因此,spPCR可以是确认在寄生线虫的功能基因组研究中的整合转基因的有用方法。 Potter和Luo(2010)包含使用spPCR检测和定位 piggyBac 转座子介导的染色体整合在果蝇中的方案,并且是我们的方法的来源类固醇。在该参考文献中描述的splinkerette和piggyBac 特异性寡核苷酸可以在没有修饰的情况下用于类花青苷。对于感兴趣的读者,可以在Viney和Lok(2007)中发现寄生线虫的生物学的一般综述,以及基于方法的文章。 stercoralis作为实验模型,具有转基因的信息,可以在Lok(2007)中找到。

关键字:粪, 转基因, 染色体整合, 转座子, splinkerette-PCR



图1.通过splinkerette PCR(改编自Potter和Luo,2010)中转基因整合的方法的图谱表示

材料和试剂

  1.  自由生活的成虫蠕虫
  2. 基因组DNA提取
    Gentra Puregene Tissue Kit(QIAGEN),包括
    1. 细胞裂解液(目录号:8304295)
    2. 蛋白沉淀溶液(目录号:8273807)
    3. DNA水合溶液(目录号:8274043)
  3. 用于消化和治疗的酶
    限制酶包括BstY I,BamH I和Bgl II(New England Biolabs) 其他包括虾碱性磷酸酶(United State Biological,目录号:P4071-05)和外切核酸酶I(New England Biolabs,目录号:M0293S)
  4. 连接试剂
    10x连接酶缓冲液和T4连接酶(New England Biolabs,目录号:M0202S)
  5. PCR试剂
    5x Phusion高保真缓冲液,Phusion HF DNA聚合酶(New England Biolabs,目录号:M0530S)
  6. 寡核苷酸和引物
    表1中详述的寡核苷酸必须合成,  
    所有序列来自Potter和Luo(2010),表S13
  7. TE缓冲液(New England Biolabs,目录号:E6293)
  8. 10x NEB缓冲区2
  9. 1%琼脂糖凝胶
    Oligo或Primer  
    序列
    SPLNK-BOT  
      5'-CGAAGAGTAACCGTTGCTAGGAGAGACCGTGGCTGAATGAGACTGGTGTCGACACTAGTGG-3'
    SPLNK-GATC-TOP   
    5'-GATCCCACTAGTGTCGACACCAGTCTCTAATTTTTTTTTTCAAAAAAA-3'
    SPLNK#1  
    5'-CGAAGAGTAACCGTTGCTAGGAGAGACC-3'
    SPLNK#2   
    5'-GTGGCTGAATGAGACTGGTGTCGAC-3'
    3'SPLNK-PB#1   
    5'-GTTTGTTGAATTTATTATTAGTATGTAAG-3'
    5'SPLNK-PB#1   
    5'-ACCGCATTGACAAGCACG-3'
    3'SPLNK-PB#2   
    5'-CGATAAAACACATGCGTC-3'
    5'SPLNK-PB#2   
    5'-CTCCAAGCGGCGACTGAG-3'
    3'SPLNK-PB-SEQ   
    5'-ACGCATGATTATCTTTAAC-3'
    5'SPLNK-PB-SEQ   
    5'-CGACTGAGATGTCCTAAATGC-3'
    表1.  用于piggyBac转座子介导的转基因整合的splinkerette PCR作图的寡核苷酸和引物

设备

  1. PCR热循环仪(Mastercycler Personal,Eppendorf)
  2. 凝胶电泳
  3. 凝胶文件(FOTODYNE,型号:FOTO/Analyst FX)

程序

  1. 从在22℃下温育48小时的新鲜宿主粪便的木炭培养物收集稳定转化的自由生活的成人 (Lok,2007) 使用Gentra Puregene Tissue Kit从约500只自由生活的成虫中提取基因组DNA(gDNA)。 寄生虫不需要均质化。 将分离的蠕虫以14,000×g离心,然后在步骤2中代替"研磨的组织",然后用Gentra Puregene Tissue Kit的制造商方案的步骤2b,使用推荐用于组织样品的试剂体积 5-10 mg。 将提取的gDNA重悬于50μlTE缓冲液(10mM Tris-HCl,1mM EDTA,pH 8.0)中,得到约25ng /μl。
  2. 用BstYI,BamHI和Bgl II消化基因组DNA(〜100ng)2小时,每个反应的总体积为15μl(图1A)。按照制造商对每种限制酶的反应条件和失活步骤的说明。
  3. 在100μl的总反应体积中加入5μl100μMSPLINK-BOT寡核苷酸,5μl100μMSPLINK-GATC-TOP寡核苷酸和10×NEB缓冲液2。加热至95℃3分钟,然后在台上冷却至室温,以退火分散体寡核苷酸。使用T4连接酶在16℃下在总体积30μl中用G连接酶将消化的gDNA(具有GATC粘性末端)与退火的分离寡核苷酸一起温育8小时(图1B)。
  4. 在将基因组DNA限制性片段连接到splinkerette寡核苷酸后进行两轮spPCR。
    1. 对于第一轮spPCR,组合引物SPLINK#1,其靶向  连接的splinkerette寡核苷酸,用引物3'SPLNK-PB#1 或引物5'SPLNK-PB#1。 3'SPLNK-PB#1和5'SPLNK-PB#1靶向3'和3' 5'末端。进行 用Phusion聚合酶和5-20ng的扩增 splinkerette连接的基因组DNA作为模板。热循环条件 是:
      1. 98℃1分钟
      2. 98℃20秒
      3. 55℃15秒
      4. 72℃2分钟
      5. 29个循环从ii到iv
      6. 最后在72℃延伸10分钟。
    2. 第二轮spPCR是使用与引物3'SPLNK-PB#2或引物5'SPLNK-PB#2配对的引物SPLNK#2和来自第一轮spPCR的spPCR产物的1:20稀释液进行的巢式PCR作为模板。使用与第一轮spPCR所指定的相同的热循环条件,用Phusion高保真DNA聚合酶进行第二轮扩增。通过1%琼脂糖凝胶电泳分析所有PCR产物。
    3. 孵育第二轮spPCR的产品与1微升虾碱 磷酸酶(1U/ml)和1μl外切核酸酶I(20,000U/ml)在37℃下2  h,然后在80℃下15分钟(图1C)。
    4. 使用引物3'SPLNK-PB-SEQ ...直接序列处理的PCR产物 和5'SPLNK-PB-SEQ,其靶向转基因的3'和5'端 序列。识别整合的染色体位置 基于产生的序列和基因组中的匹配片段 数据库(图1D,1E)。

致谢

该协议详述了最初在Shao等人(2012)中公开的方法。 这项工作由来自美国国立卫生研究院的授权号AI050668资助。

参考文献

  1. Lok,J.B。 Strongyloides stercoralis :用于寄生线虫生物学的翻译研究的模型(2007年2月17日), WormBook C. elegans 研究社群,WormBook,doi/10.1895/wormbook.1.134.1, http: //www.wormbook.org。
  2. Potter,C.J。和Luo,L。(2010)。 Splinkerette PCR用于在果蝇中映射转座元件。 < em> PLoS One 5(4): e10168。 
  3. Shao,H.,Li,X.,Nolan,T.J.,Massey,H.C.,Jr.,Pearce,E.J.and Lok,J.B。(2012)。 转座子介导的转基因在寄生线虫中的染色体整合 Strongyloides ratti 和 建立稳定的转基因株系。 PLoS Pathog 8(8):e1002871。
  4. Viney,M.E.and Lok,J.B。 Strongyloides (2007年5月23日), WormBook C。 elegans Research Community,WormBook,doi/10.1895/wormbook.1.141.1, http://www.wormbook。 org
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Shao, H. and Lok, J. B. (2014). Detection of piggyBac-mediated Transposition by Splinkerette PCR in Transgenic Lines of Strongyloides ratti. Bio-protocol 4(1): e1015. DOI: 10.21769/BioProtoc.1015.
  2. Shao, H., Li, X., Nolan, T. J., Massey, H. C., Jr., Pearce, E. J. and Lok, J. B. (2012). Transposon-mediated chromosomal integration of transgenes in the parasitic nematode Strongyloides ratti and establishment of stable transgenic lines. PLoS Pathog 8(8): e1002871.
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