Abstract
To construct an effective site-specific integration system in the silkworm, we examined if phiC31 integrase works in silkworm embryos. As an assay system, we constructed an extrachromosomal cassette exchange reaction system between two attP sites of an acceptor plasmid and two attB sites of a donor plasmid. To evaluate the activity, integrase mRNAs synthesized from three different plasmids were used. We injected a mixture of the acceptor and donor plasmids with the mRNA synthesized in vitro from one of the three plasmids into silkworm embryos at 4–6 h after oviposition and recovered plasmid DNAs from the embryos 3 days after injection. The resultant plasmids were transformed into Escherichia coli and spread on selection medium plates containing the appropriate antibiotics. A colony-forming assay and restriction enzyme digestion of the plasmids purified from the colonies showed that the phiC31 integrase worked very efficiently in the silkworm embryos. Notably, a phiC31 integrase mRNA synthesized from two of the plasmids produced cassette exchange plasmids at a high frequency, suggesting that the mRNA can be used to construct a targeted integration system in silkworms.
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References
Bateman JR, Lee AM, Wu CT (2006) Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange. Genetics 173:769–777
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
Fish MP, Groth AC, Calos MP, Nusse R (2007) Creating transgenic Drosophila by microinjecting the site-specific phiC31 integrase mRNA, a transgene-containing donor plasmid. Nat Protoc 2:2325–2331
Groth AC, Olivares EC, Thyagarajan B, Calos MP (2000) A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci USA 97:5995–6000
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
Horn C, Handler AM (2005) Site-specific genomic targeting in Drosophila. Proc Natl Acad Sci USA 102:12483–12488
Imamura M, Nakai J, Inoue S, Quan GX, Kanda T, Tamura T (2003) Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori. Genetics 165:1329–1340
Inoue S, Kanda T, Imamura M, Quan GX, Kojima K, Tanaka H, Tomita M, Hino R, Yoshizato K, Mizuno S, Tamura T (2005) A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins. Insect Biochem Mol Biol 35:51–59
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
Labbe GM, Nimmo DD, Alphey L (2011) piggybac- and PhiC31-mediated genetic transformation of the Asian tiger mosquito, Aedes albopictus (Skuse). PLoS Negl Trop Dis 4:e788
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
Nakayama G, Kawaguchi Y, Koga K, Kusakabe T (2006) Site-specific gene integration in cultured silkworm cells mediated by phiC31 integrase. Mol Genet Genomics 275:1–8
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
Quan GX, Kobayashi I, Kojima K, Uchino K, Kanda T, Sezutsu H, Shimada T, Tamura T (2007) Rescue of white egg 1 mutant by introduction of the wild-type Bombyx kynurenine 3-monooxygenase gene. Insect Sci 14:85–92
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
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
Shimizu K, Kamba M, Sonobe H, Kanda T, Klinakis AG, Savakis C, Tamura T (2000) Extrachromosomal transposition of the transposable element Minos occurs in embryos of the silkworm Bombyx mori. Insect Mol Biol 9:277–281
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 (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18:81–84
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
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
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
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
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
Tomita M, Munetsuna H, Sato T, Adachi T, Hino R, Hayashi M, Shimizu K, Nakamura N, Tamura T, Yoshizato K (2003) Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nat Biotechnol 21:52–56
Tomita M, Hino R, Ogawa S, Iizuka M, Adachi T, Shimizu K, Sotoshiro H, Yoshizato K (2007) A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon. Transgenic Res 16:449–465
Uchino K, Imamura M, Sezutsu H, Kobayashi I, Kojima K, Kanda T, Tamura T (2006) Evaluating promoter sequences for trapping an enhancer activity in the silkworm Bombyx mori. J Insect Biotechnol Sericol 75:89–97
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
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
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
Acknowledgments
We thank Mr. Kaoru Nakamura and Mr. Koji Hashimoto for rearing the silkworms. This work was supported by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan.
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Communicated by T. Clandinin.
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Yonemura, N., Tamura, T., Uchino, K. et al. PhiC31 integrase-mediated cassette exchange in silkworm embryos. Mol Genet Genomics 287, 731–739 (2012). https://doi.org/10.1007/s00438-012-0711-y
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DOI: https://doi.org/10.1007/s00438-012-0711-y