采用Splinkerette PCR检测类圆线虫转基因株系中的piggyBac介导转位

Hongguang Shao; James B. Lok

doi:10.21769/BioProtoc.1015

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Peer-reviewed

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

HS Hongguang Shao

JL James B. Lok email

发布: 2014年01月05日第4卷第1期 DOI: 10.21769/BioProtoc.1015 浏览次数: 14661

评审: Fanglian He

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Aug 2012

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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

Free-living adult worms
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 )
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 )
Ligation reagents
10x Ligase buffer and T4 Ligase (New England Biolabs, catalog number: M0202S )
PCR reagents
5x Phusion High-Fidelity Buffer, Phusion HF DNA Polymerase (New England Biolabs, catalog number: M0530S )
Oligonucleotides and primers
The oligonucleotides detailed in Table 1 must be synthesized
All sequences are from Potter and Luo (2010), Table S13
TE buffer (New England Biolabs, catalog number: E6293 )
10x NEB Buffer 2

1% agarose gel

Table 1. Oligonucleotides and primers for splinkerette PCR mapping of piggyBac transposon-mediated transgene integrations

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’

Equipment

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

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

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.
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.