Skip to main content
SpringerLink
Log in

Cre-mediated targeted gene activation in the middle silk glands of transgenic silkworms (Bombyx mori)

  • Original Paper
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

Cre-mediated recombination is widely used to manipulate defined genes spatiotemporally in vivo. The present study evaluated the Cre/loxP system in Bombyx mori by establishing two transgenic lines. One line contained a Cre recombinase gene controlled by a sericin-1 gene (Ser1) promoter. The other line contained a loxP-Stop-loxP-DsRed cassette driven by the same Ser1 promoter. The precise deletion of the Stop fragment was found to be triggered by Cre-mediated site-specific excision, and led to the expression of DsRed fluorescence protein in the middle silk glands of all double-transgenic hybrids. This result was also confirmed by phenotypical analysis. Hence, the current study demonstrated the feasibility of Cre-mediated site-specific recombination in B. mori, and opened a new window for further refining genetic tools in silkworms.

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
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

Ser1 :

Sericin-1 gene

floxed:

Flanked by two loxP sites

RFP:

DsRed fluorescence protein

pA:

SV40 polyadenylation signal

AMSG:

Anterior subpart of middle silk gland

MMSG:

Middle subpart of middle silk gland

PMSG:

Posterior subpart of middle silk gland

MSG:

Middle silk gland

PSG:

Posterior silk gland

References

  • Albert H, Dale EC, Lee E, Ow DW (1995) Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J 7(4):649–659

    Article  PubMed  CAS  Google Scholar 

  • Branda CS, Dymecki SM (2004) Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev Cell 6(1):7–28. doi:S153458070300399X

    Google Scholar 

  • Buchholz F, Ringrose L, Angrand PO, Rossi F, Stewart AF (1996) Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination. Nucleic Acids Res 24(21):4256–4262. doi:10.1093/nar/24.21.4256

    Article  PubMed  CAS  Google Scholar 

  • Gage LP (1974) Polyploidization of the silk gland of Bombyx mori. J Mol Biol 86(1):97–108

    Article  PubMed  CAS  Google Scholar 

  • Garrick D, Fiering S, Martin DI, Whitelaw E (1998) Repeat-induced gene silencing in mammals. Nat Genet 18(1):56–59. doi:10.1038/ng0198-56

    Article  PubMed  CAS  Google Scholar 

  • Golic KG, Golic MM (1996) Engineering the Drosophila genome: chromosome rearrangements by design. Genetics 144(4):1693–1711

    PubMed  CAS  Google Scholar 

  • Gong WJ, Golic KG (2003) Ends-out, or replacement, gene targeting in Drosophila. Proc Natl Acad Sci USA 100(5):2556–2561. doi:10.1073/pnas.0535280100

    Article  PubMed  CAS  Google Scholar 

  • Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K (1994) Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265(5168):103–106

    Article  PubMed  CAS  Google Scholar 

  • Herault Y, Rassoulzadegan M, Cuzin F, Duboule D (1998) Engineering chromosomes in mice through targeted meiotic recombination (TAMERE). Nat Genet 20(4):381–384. doi:10.1038/3861

    Article  PubMed  CAS  Google Scholar 

  • Herrera PL (2000) Adult insulin- and glucagon-producing cells differentiate from two independent cell lineages. Development 127(11):2317–2322

    PubMed  CAS  Google Scholar 

  • Hoa TT, Bong BB, Huq E, Hodges TK (2002) Cre/lox site-specific recombination controls the excision of a transgene from the rice genome. Theor Appl Genet 104(4):518–525. doi:10.1007/s001220100748

    Article  PubMed  CAS  Google Scholar 

  • Horn C, Wimmer EA (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210(12):630–637

    Article  PubMed  CAS  Google Scholar 

  • 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(3):1329–1340

    PubMed  CAS  Google Scholar 

  • Indra AK, Warot X, Brocard J, Bornert JM, Xiao JH, Chambon P, Metzger D (1999) Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res 27(22):4324–4327. doi:10.1093/nar/27.22.4324

    Article  PubMed  CAS  Google Scholar 

  • Isobe R, Kojima K, Matsuyama T, Quan GX, Kanda T, Tamura T, Sahara K, Asano SI, Bando H (2004) Use of RNAi technology to confer enhanced resistance to BmNPV on transgenic silkworms. Arch Virol 149(10):1931–1940

    PubMed  CAS  Google Scholar 

  • Kozak M (1991) Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 266(30):19867–19870

    PubMed  CAS  Google Scholar 

  • Lakso M, Sauer B, Mosinger B Jr, Lee EJ, Manning RW, Yu SH, Mulder KL, Westphal H (1992) Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci USA 89(14):6232–6236

    Article  PubMed  CAS  Google Scholar 

  • Lam KP, Rajewsky K (1998) Rapid elimination of mature autoreactive B cells demonstrated by Cre-induced change in B cell antigen receptor specificity in vivo. Proc Natl Acad Sci USA 95(22):13171–13175

    Article  PubMed  CAS  Google Scholar 

  • Liu Y, Yu L, Guo X, Guo T, Wang S, Lu C (2006) Analysis of tissue-specific region in sericin 1 gene promoter of Bombyx mori. Biochem Biophys Res Commun 342(1):273–279. doi:S0006-291X(06)00236-1

    Google Scholar 

  • Matsuno K, Hui CC, Takiya S, Suzuki T, Ueno K, Suzuki Y (1989) Transcription signals and protein binding sites for sericin gene transcription in vitro. J Biol Chem 264(31):18707–18713

    PubMed  CAS  Google Scholar 

  • Nagy A, Moens C, Ivanyi E, Pawling J, Gertsenstein M, Hadjantonakis AK, Pirity M, Rossant J (1998) Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles. Curr Biol 8(11):661–664. doi:S0960-9822(98)70254-4

    Google Scholar 

  • Nakayama G, Kawaguchi Y, Koga K, Kusakabe T (2006) Site-specific gene integration in cultured silkworm cells mediated by φC31 integrase. Mol Genet Genomics 275(1):1–8. doi:10.1007/s00438-005-0026-3

    Article  PubMed  CAS  Google Scholar 

  • O’Gorman S, Fox DT, Wahl GM (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251(4999):1351–1355

    Article  PubMed  Google Scholar 

  • Perdrix-Gillot S (1979) DNA synthesis and endomitoses in the giant nuclei of the silkgland of Bombyx mori. Biochimie 61(2):171–204

    Article  PubMed  CAS  Google Scholar 

  • Quan GX, Kanda T, Tamura T (2002) Induction of the white egg 3 mutant phenotype by injection of the double-stranded RNA of the silkworm white gene. Insect Mol Biol 11(3):217–222. doi:10.1046/j.13652583.2002.00328.x

    Article  PubMed  CAS  Google Scholar 

  • Rong YS, Golic KG (2001) A targeted gene knockout in Drosophila. Genetics 157(3):1307–1312

    PubMed  CAS  Google Scholar 

  • Sauer B (1987) Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol Cell Biol 7(6):2087–2096

    PubMed  CAS  Google Scholar 

  • Suzuki MG, Funaguma S, Kanda T, Tamura T, Shimada T (2003) Analysis of the biological functions of a doublesex homologue in Bombyx mori. Dev Genes Evol 213(7):345–354. doi:10.1007/s00427-003-0334-8

    Article  PubMed  CAS  Google Scholar 

  • Tabunoki H, Higurashi S, Ninagi O, Fujii H, Banno Y, Nozaki M, Kitajima M, Miura N, Atsumi S, Tsuchida K, Maekawa H, Sato R (2004) A carotenoid-binding protein (CBP) plays a crucial role in cocoon pigmentation of silkworm (Bombyx mori) larvae. FEBS Lett 567(2–3):175–178. doi:10.1016/j.febslet.2004.04.067

    Article  PubMed  CAS  Google Scholar 

  • Takasu Y, Hata T, Uchino K, Zhang Q (2010) Identification of Ser2 proteins as major sericin components in the non-cocoon silk of Bombyx mori. Insect Biochem Mol Biol 40(4):339–344. doi:S0965-1748(10)00039-1

  • 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(1):81–84. doi:10.1038/71978

    Article  PubMed  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(33):11751–11756. doi:10.1073/pnas.0500954102

    Google Scholar 

  • Tanimoto K, Liu Q, Bungert J, Engel JD (1999) Effects of altered gene order or orientation of the locus control region on human beta-globin gene expression in mice. Nature 398(6725):344–348. doi:10.1038/18698

    Article  PubMed  CAS  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(3):473–487. doi:10.1007/s11248-009-9328-2

    Article  PubMed  CAS  Google Scholar 

  • Tateno M, Toyooka M, Shikano Y, Takeda S, Kuwabara N, Sezutsu H, Tamura T (2009) Production and characterization of the recombinant human μ-opioid receptor from transgenic silkworms. J Biochem 145(1):37–42. doi:10.1093/jb/mvn147

    Article  PubMed  CAS  Google Scholar 

  • Thummel R, Burket CT, Brewer JL, Sarras MP Jr, Li L, Perry M, McDermott JP, Sauer B, Hyde DR, Godwin AR (2005) Cre-mediated site-specific recombination in zebrafish embryos. Dev Dyn 233(4):1366–1377. doi:10.1002/dvdy.20475

    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(3):371–377

    Google Scholar 

  • Tsien JZ, Chen DF, Gerber D, Tom C, Mercer EH, Anderson DJ, Mayford M, Kandel ER, Tonegawa S (1996) Subregion- and cell type-restricted gene knockout in mouse brain. Cell 87(7):1317–1326. doi:S0092-8674(00)81826-7

    Google Scholar 

  • Utomo AR, Nikitin AY, Lee WH (1999) Temporal, spatial, and cell type-specific control of Cre-mediated DNA recombination in transgenic mice. Nat Biotechnol 17(11):1091–1096. doi:10.1038/15073

    Article  PubMed  CAS  Google Scholar 

  • Vergunst AC, Jansen LE, Fransz PF, de Jong JH, Hooykaas PJ (2000) Cre/lox-mediated recombination in Arabidopsis: evidence for transmission of a translocation and a deletion event. Chromosoma 109(4):287–297

    Article  PubMed  CAS  Google Scholar 

  • Xie HB, Golic KG (2004) Gene deletions by ends-in targeting in Drosophila melanogaster. Genetics 168(3):1477–1489. doi:10.1534/genetics.104.030882

    Article  PubMed  CAS  Google Scholar 

  • Zhao A, Zhao T, Zhang Y, Xia Q, Lu C, Zhou Z, Xiang Z, Nakagaki M (2009) New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm. Transgenic Res 19(1):29–44. doi:10.1007/s11248-009-9295-7

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Dr. Pedro Luis Herrera for pBS302 plasmid, Dr. Wen-Hwa Lee for pAU8-5 plasmid, and Dr. Hongjuan Cui and Dr. Shiping Liu for their critical comments on the manuscript. We also thank the Editor and reviewers for their constructive comments. The work was supported by the National Basic Research Program of China (2012CB114600), the National Hi-Tech Research and Development Program of China (2011AA100306), the National Natural Science Foundation of China (31000981) and the Graduate Innovation Foundation of Southwest University (ky2009002, kb2010002 and kb2011008).

Author information

Authors and Affiliations

  1. State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400715, China

    Jianping Duan, Hanfu Xu, Sanyuan Ma, Huizhen Guo, Feng Wang, Ping Zhao & Qingyou Xia

Authors
  1. Jianping Duan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hanfu Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sanyuan Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Huizhen Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Feng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ping Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Qingyou Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Qingyou Xia.

Additional information

Jianping Duan and Hanfu Xu contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Duan, J., Xu, H., Ma, S. et al. Cre-mediated targeted gene activation in the middle silk glands of transgenic silkworms (Bombyx mori). Transgenic Res 22, 607–619 (2013). https://doi.org/10.1007/s11248-012-9677-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-012-9677-0

Keywords

Search

Navigation