Abstract
The piggyBac transposon has been long used to integrate foreign DNA into insect genomes. However, undesirable transgene expression can result from random insertions into the genome. In this study, the efficiency of chimeric Gal4-piggyBac transposase in directing integration onto a DNA target plasmid was evaluated in cultured silkworm Bombyx mori Bm-12 and fruit fly Drosophila Schneider 2 (S2) cells. The Gal4-piggyBac transposase has a Gal4 DNA-binding domain (DBD), and the target plasmid has upstream activating sequences (UAS) to which the Gal4 DBD can bind with high affinity. The results indicate that, in the Bm-12 and S2 cells, transpositional activity of Gal4-piggyBac transposase was increased by 4.0 and 7.5 times, respectively, compared to controls, where Gal4-UAS interaction was absent. Moreover, the Gal4-piggyBac transposase had the ability of directing piggyBac element integration to certain sites of the target plasmid, although the target-directing specificity was not as high as expected. The chimeric piggyBac transposase has the potential for use in site-directed transgenesis and gene function research in B. mori.
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Coates, C.J., Kaminski, J.M., Summers, J.B., Segal, D.J., Miller, A.D., Kolb, A.F., 2005. Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools. Trends Biotechnol., 23(8):407–419. [doi:10.1016/j.tibtech.2005.06.009]
Dai, H., Ma, L., Wang, J., Jiang, R., Wang, Z., Fei, J., 2008. Knockdown of ecdysis-triggering hormone gene with a binary UAS/GAL4 RNA interference system leads to lethal ecdysis deficiency in silkworm. Acta Biochim. Biophys. Sin., 40(9):790–795. [doi:10.1111/j.1745-7270.2008.00460.x]
Demattei, M.V., Thomas, X., Carnus, E., Augé-Gouillou, C., Renault, S., 2010. Site-directed integration of transgenes: transposons revisited using DNA-binding-domain technologies. Genetica, 138(5):531–540. [doi:10.1007/s10709-009-9390-y]
Imamura, M., Nakai, J., Inoue, S., Quan, G.X., Kanda, T., Tamura, T., 2003. Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori. Genetics, 165(3):1329–1340.
Kaminski, J.M., Huber, M.R., Summers, J.B., Ward, M.B., 2002. Design of a nonviral vector for site-selective, efficient integration into the human genome. FASEB J., 16(10):1242–1247. [doi:10.1096/fj.02-0127hyp]
Khurad, A.M., Zhang, M.J., Deshmukh, C.G., Bahekar, R.S., Tiple, A.D., Zhang, C.X., 2009. A new continuous cell line from larval ovaries of silkworm, Bombyx mori. In Vitro Cell. Dev. Biol. Anim., 45(8):414–419. [doi:10.1007/s11626-009-9197-2]
Klueg, K.M., Alvarado, D., Muskavitch, M.A.T., Duffy, J.B., 2002. Creation of a GAL4/UAS-coupled inducible gene expression system for use in Drosophila cultured cell lines. Genesis, 34(1–2):119–122. [doi:10.1002/gene.10148]
Maragathavally, K.J., Kaminski, J.M., Coates, C.J., 2006. Chimeric Mos1 and piggyBac transposases result in sitedirected integration. FASEB J., 20(11):1880–1882. [doi:10.1096/fj.05-5485fje]
Mita, K., Kasahara, M., Sasaki, S., Nagayasu, Y., Yamada, T., Kanamori, H., Namiki, N., Kitagawa, M., Yamashita, H., Yasukochi, Y., et al., 2004. The genome sequence of silkworm, Bombyx mori. DNA Res., 11(1):27–35. [doi:10.1093/dnares/11.1.27]
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]
Nicholson, L., Singh, G.K., Osterwalder, T., Roman, G.W., Davis, R.L., Keshishian, H., 2008. Spatial and temporal control of gene expression in Drosophila using the inducible GeneSwitch GAL4 system. I. Screen for larval nervous system drivers. Genetics, 178(1):215–234. [doi:10.1534/genetics.107.081968]
Phelps, C.B., Brand, A.H., 1998. Ectopic gene expression in Drosophila using Gal4 system. Methods, 14(4):367–379. [doi:10.1006/meth.1998.0592]
Robertson, L.K., Dey, B.K., Campos, A.R., Mahaffey, J.W., 2002. Expression of the Drosophila gene disconnected using the UAS/GAL4 system. Genesis, 34(1–2):103–106. [doi:10.1002/gene.10123]
SPSS Inc., 1999. SPSS Base 9.0 Applications Guide. Chicago, Illinois, USA.
Tamura, T., Thilbert, C., Royer, C., Kanda, T., Abraham, E., Kamba, M., Kômoto, N., Thomas, J.L., Mauchamp, B., Chavancy, G., et al., 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]
The International Silkworm Genome Consortium, 2008. The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect Biochem. Mol. Biol., 38(12): 1036–1045. [doi:10.1016/j.ibmb.2008.11.004]
Thibault, S.T., Luu, H.T., Vann, N., Miller, T.A., 1999. Precise excision and transposition of piggyBac in pink bollworm embryos. Insect Mol. Biol., 8(1):119–123. [doi:10.1046/j.1365-2583.1999.810119.x]
Wilson, M.H., Coates, C.J., George, A.L.Jr., 2007. piggyBac transposon-mediated gene transfer in human cells. Mol. Ther., 15(1):139–145. [doi:10.1038/sj.mt.6300028]
Wu, S.C.Y., Meir, Y.J.J., Coates, C.J., Handler, A.M., Pelczar, P., Moisyadi, S., Kaminski, J.M., 2006. piggyBac is a flexible and highly active transposon as compared to Sleeping Beauty, Tol2 and Mos1 in mammalian cells. PNAS, 103(41):15008–15013. [doi:10.1073/pnas.0606979103]
Wu, S.C.Y., Maragathavally, K.J., Coates, C.J., Kaminski, J.M., 2007. Steps Toward Targeted Insertional Mutagenesis with Class II Transposable Elements. In: Davis, G., Kayser, K.J. (Eds.), Methods in Molecular Biology. Vol. 435: Chromosomal Mutagenesis. Human Press Inc., Totowa, NJ, p.139–151.
Xia, Q.Y., Zhou, Z.Y., Lu, C., Cheng, D.J., Dai, F.Y., Li, B., Zhao, P., Zha, X.F., Cheng, T.C., Chai, C.L., et al., 2004. A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science, 306(5703):1937–1940. [doi:10.1126/science.1102210]
Xue, G.P., Johnson, J.S., Dalrymple, B.P., 1999. High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. J. Microbiol. Meth., 34(3):183–191. [doi:10.1016/S0167-7012(98)00087-6]
Yamagata, T., Sakurai, T., Uchino, K., Sezutsu, H., Tamura, T., Kanzaki, R., 2008. GFP labeling of neurosecretory cells with the GAL4/UAS system in the silkmoth brain enables selective intracellular staining of neurons. Zool. Sci., 25(5):509–516. [doi:10.2108/zsj.25.509]
Zhong, J., Yedvobnick, B., 2009. Targeted gain-of-function screening in Drosophila using GAL4-UAS and random transposon insertions. Genet. Res., 91(4):243–258. [doi:10.1017/S0016672309990152]
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Project supported by the National Basic Research Program (973) of China (No. 2009CB119200) and the Natural Science Foundation of Zhejiang Province (No. Y304122), China
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Wang, N., Jiang, Cy., Jiang, Mx. et al. Using chimeric piggyBac transposase to achieve directed interplasmid transposition in silkworm Bombyx mori and fruit fly Drosophila cells. J. Zhejiang Univ. Sci. B 11, 728–734 (2010). https://doi.org/10.1631/jzus.B1000139
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DOI: https://doi.org/10.1631/jzus.B1000139