Skip to main content
SpringerLink
Log in

phiC31-integrase-mediated, site-specific integration of transgenes in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

  • Original Research Paper
  • Published:
Applied Entomology and Zoology Aims and scope Submit manuscript

Abstract

Transgenic silkworms can be useful for investigating the functions of genes in the post-genomic era. However, the common method of using a transposon as an insertion tool may result in the random integration of a foreign gene into the genome and suffer from a strong position effect. To overcome these problems, it is necessary to develop a site-specific integration system. It is known that phiC31 integrase has the capacity to mediate recombination between the target sequences attP and attB. To test the availability of site-specific integration in the silkworm, we first examined the efficiency of recombination between the target sites of the two plasmids in silkworm embryos and found that the frequency of recombination was very high. Then we constructed a host strain that possessed the target sequence attP using the common method. We injected the donor plasmid together with the phiC31 integrase mRNA into the embryos of the host strain and obtained positive lines. Structural analysis of the lines showed that site-specific integration occurred by recombination between the genomic attP site and the attB site of the donor plasmid. We can conclude from the results that phiC31 integrase has the ability to mediate the site-specific integration of transgenes into the silkworm chromosome.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Atsumi S, Miyamoto K, Yamamoto K, Narukawa J, Kawai S, Sezutsu H, Kobayashi I, Uchino K, Tamura T, Mita K, Kadono-Okuda K, Wada S, Kanda K, Goldsmith MR, Noda H (2012) Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori. Proc Natl Acad Sci USA 109:E1591–E1598. doi:10.1073/pnas.1120698109

    Article  PubMed  CAS  Google Scholar 

  • Bateman JR, Lee AM, Wu CT (2006) Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange. Genetics 173:769–777. doi:10.1534/genetics.106.056945

    Article  PubMed  CAS  Google Scholar 

  • Bischof J, Maeda RK, Hediger M, Karch F, Basler K (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci USA 104:3312–3317. doi:10.1073/pnas.0611511104

    Article  PubMed  CAS  Google Scholar 

  • Consortium ISG (2008) The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect Biochem Mol Biol 38:1036–1045. doi:10.1016/j.ibmb.2008.11.004

    Article  Google Scholar 

  • Fish MP, Groth AC, Calos MP, Nusse R (2007) Creating transgenic Drosophila by microinjecting the site-specific phiC31 integrase mRNA and a transgene-containing donor plasmid. Nat Protoc 2:2325–2331. doi:10.1038/nprot.2007.328

    Article  PubMed  CAS  Google Scholar 

  • Groth AC, Fish M, Nusse R, Calos MP (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166:1775–1782. doi:10.1534/genetics.166.4.1775

    Article  PubMed  CAS  Google Scholar 

  • Horn C, Handler AM (2005) Site-specific genomic targeting in Drosophila. Proc Natl Acad Sci USA 102:12483–12488. doi:10.1073/pnas.0504305102

    Article  PubMed  CAS  Google Scholar 

  • Horn C, Schmid BG, Pogoda FS, Wimmer EA (2002) Fluorescent transformation markers for insect transgenesis. Insect Biochem Mol Biol 32:1221–1235. doi:org/10.1016/S0965-1748(02)00085-1

    Article  PubMed  CAS  Google Scholar 

  • Ishikawa Y, Tanaka N, Murakami K, Uchiyama T, Kumaki S, Tsuchiya S, Kugoh H, Oshimura M, Calos MP, Sugamura K (2006) Phage phiC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines. J Gene Med 8:646–653. doi:10.1002/jgm.891

    Article  PubMed  CAS  Google Scholar 

  • Ito K, Kidokoro K, Sezutsu H, Nohata J, Yamamoto K, Kobayashi I, Uchino K, Kalyebi A, Eguchi R, Hara W, Tamura T, Katsuma S, Shimada T, Mita K, Kadono-Okuda K (2008) Deletion of a gene encoding an amino acid transporter in the midgut membrane causes resistance to a Bombyx parvo-like virus. Proc Natl Acad Sci USA 105:7523–7527. doi:10.1073/pnas.0711841105

    Google Scholar 

  • Keravala A, Lee S, Thyagarajan B, Olivares EC, Gabrovsky VE, Woodard LE, Calos MP (2009) Mutational derivatives of PhiC31 integrase with increased efficiency and specificity. Mol Ther 17:112–120. doi:10.1038/mt.2008.241

    Article  PubMed  CAS  Google Scholar 

  • Labbe GM, Nimmo DD, Alphey L (2010) Piggybac- and PhiC31-mediated genetic transformation of the Asian tiger mosquito, Aedes albopictus (Skuse). PLoS Negl Trop Dis 4:e788. doi:10.1371/journal.pntd.0000788

    Article  PubMed  Google Scholar 

  • Ma S, Zhang S, Wang F, Liu Y, Xu H, Liu C, Lin Y, Zhao P, Xia Q (2012) Highly efficient and specific genome editing in silkworm using custom TALENs. PLoS ONE 7:e45035. doi:10.1371/journal.pone.0045035

    Article  PubMed  CAS  Google Scholar 

  • Meredith JM, Basu S, Nimmo DD, Larget-Thiery I, Warr EL, Underhill A, McArthur CC, Carter V, Hurd H, Bourgouin C, Eggleston P (2011) Site-specific integration and expression of an anti-malarial gene in transgenic Anopheles gambiae significantly reduces Plasmodium infections. PLoS ONE 6:e14587. doi:10.1371/journal.pone.0014587

  • Nimmo DD, Alphey L, Meredith JM, Eggleston P (2006) High efficiency site-specific genetic engineering of the mosquito genome. Insect Mol Biol 15:129–136. doi:10.1111/j.1365-2583.2006.00615.x

    Article  PubMed  CAS  Google Scholar 

  • Sajwan S, Takasu Y, Tamura T, Uchino K, Sezutsu H, Zurovec M (2013) Efficient disruption of endogenous Bombyx gene by TAL effector nucleases. Insect Biochem Mol Biol 43:17–23. doi:10.1016/j.ibmb.2012.10.011

    Google Scholar 

  • Sakudoh T, Sezutsu H, Nakashima T, Kobayashi I, Fujimoto H, Uchino K, Banno Y, Iwano H, Maekawa H, Tamura T, Kataoka H, Tsuchida K (2007) Carotenoid silk coloration is controlled by a carotenoid-binding protein, a product of the Yellow blood gene. Proc Natl Acad Sci USA 104:8941–8946. doi:10.1073/pnas.0702860104

    Google Scholar 

  • Schetelig MF, Scolari F, Handler AM, Kittelmann S, Gasperi G, Wimmer EA (2009) Site-specific recombination for the modification of transgenic strains of the Mediterranean fruit fly Ceratitis capitata. Proc Natl Acad Sci USA 106:18171–18176. doi:10.1073/pnas.0907264106

    Article  PubMed  CAS  Google Scholar 

  • Takasu Y, Kobayashi I, Beumer K, Uchino K, Sezutsu H, Sajwan S, Carroll D, Tamura T, Zurovec M (2010) Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection. Insect Biochem Mol Biol 40:759–765. doi:10.1016/j.ibmb.2010.07.012

    Article  PubMed  CAS  Google Scholar 

  • Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P, Toshiki T, Chantal T, Corinne R, Toshio K, Eappen A, Mari K, Natuo K, Jean-Luc T, Bernard M, Gerard C, Paul S, Malcolm F, Jean-Claude P, Pierre C (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18:81–84. doi:10.1038/71978

    Article  PubMed  CAS  Google Scholar 

  • Tamura T, Kuwabara K, Uchino K, Kobayashi I, Kanda T (2007) An improved DNA injection method for silkworm eggs drastically increases the efficiency of producing transgenic silkworms. J Insect Biotechnol Sericol 76:155–159

    CAS  Google Scholar 

  • Tan A, Tanaka H, Tamura T, Shiotsuki T (2005) Precocious metamorphosis in transgenic silkworms overexpressing juvenile hormone esterase. Proc Natl Acad Sci USA 102:11751–11756. doi:10.1073/pnas.0500954102

    Article  PubMed  CAS  Google Scholar 

  • Tatemastu K, Sezutsu H, Tamura T (2012) Utilization of transgenic silkworms for recombinant protein production. J Biotechnol Biomaterial S9:004. doi:10.4172/2155-952X.S9-004

    Google Scholar 

  • Tatematsu K, Kobayashi I, Uchino K, Sezutsu H, Iizuka T, Yonemura N, Tamura T (2010) Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori. Transgenic Res 19:473–487. doi:10.1007/s11248-009-9328-2

    Article  PubMed  CAS  Google Scholar 

  • Thyagarajan B, Olivares EC, Hollis RP, Ginsburg DS, Calos MP (2001) Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Mol Cell Biol 21:3926–3934. doi:10.1128/MCB.21.12.3926-3934.2001

    Article  PubMed  CAS  Google Scholar 

  • Tomita M (2011) Transgenic silkworms that weave recombinant proteins into silk cocoons. Biotechnol Lett 33:645–654

    Article  PubMed  CAS  Google Scholar 

  • Tomita S, Kanda T, Imanishi S, Tamura T (1999) Yeast FLP recombinase-mediated excision in cultured cells and embryos of the silkworm, Bombyx mori (Lepidoptera: Bombycidae). Appl Entomol Zool 34:371–377. doi:10.1007/s10529-010-0498-z

    Google Scholar 

  • Uchino K, Imamura M, Shimizu K, Kanda T, Tamura T (2007) Germ line transformation of the silkworm, Bombyx mori, using the transposable element minos. Mol Genet Genomics 277:213–220. doi:10.1007/s00438-006-0176-y

    Article  PubMed  CAS  Google Scholar 

  • Uchino K, Sezutsu H, Imamura M, Kobayashi I, Tatematsu K, Iizuka T, Yonemura N, Mita K, Tamura T (2008) Construction of a piggyBac-based enhancer trap system for the analysis of gene function in silkworm Bombyx mori. Insect Biochem Mol Biol 38:1165–1173. doi:10.1016/j.ibmb.2008.09.009

    Article  PubMed  CAS  Google Scholar 

  • Venken KJ, He Y, Hoskins RA, Bellen HJ (2006) P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314:1747–1751. doi:10.1126/science.1134426

    Article  PubMed  CAS  Google Scholar 

  • Yonemura N, Tamura T, Uchino K, Kobayashi I, Tatematsu K, Iizuka T, Sezutsu H, Muthulakshmi M, Nagaraju J, Kusakabe T (2012) PhiC31 integrase-mediated cassette exchange in silkworm embryos. Mol Genet Genomics 287:731–739. doi:10.1007/s00438-012-0711-y

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Mr. Kaoru Nakamura, Mr. Toshihiko Misawa, and Mr. Koji Hashimoto for rearing the silkworms. This work was partly supported by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan.

Author information

Authors and Affiliations

  1. Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8634, Japan

    Naoyuki Yonemura, Toshiki Tamura, Keiro Uchino, Isao Kobayashi, Ken-ichiro Tatematsu, Tetsuya Iizuka, Takuya Tsubota & Hideki Sezutsu

  2. Laboratory of Molecular Genetics, Centre for DNA Fingerprinting and Diagnostics, Tuljaguda, Nampally, Hyderabad, India

    Mayandi Muthulakshmi & Javaregowda Nagaraju

  3. Laboratory of Silkworm Science, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan

    Takahiro Kusakabe

Authors
  1. Naoyuki Yonemura
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Toshiki Tamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Keiro Uchino
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Isao Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ken-ichiro Tatematsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tetsuya Iizuka
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Takuya Tsubota
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hideki Sezutsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Mayandi Muthulakshmi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Javaregowda Nagaraju
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Takahiro Kusakabe
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Toshiki Tamura.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yonemura, N., Tamura, T., Uchino, K. et al. phiC31-integrase-mediated, site-specific integration of transgenes in the silkworm, Bombyx mori (Lepidoptera: Bombycidae). Appl Entomol Zool 48, 265–273 (2013). https://doi.org/10.1007/s13355-013-0182-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13355-013-0182-6

Keywords

Search

Navigation