Effective Expression-Independent Gene Trapping and Mutagenesis Mediated by Sleeping Beauty Transposon

Guili Song; Qing Li; Yong Long; Perry B. Hackett; Zongbin Cui

doi:10.1016/j.jgg.2012.05.010

Volume 39 Issue 9

Sep. 2012

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Article Navigation > Journal of Genetics and Genomics > 2012 > 39(9): 503-520

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Effective Expression-Independent Gene Trapping and Mutagenesis Mediated by Sleeping Beauty Transposon

doi: 10.1016/j.jgg.2012.05.010

a
The Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, 7 Donghu Rd., Wuhan, Hubei 430072, China
b
Graduate University of the Chinese Academy of Sciences, Beijing 100049, China
c
Genetics, Cell Biology, and Development, University of Minnesota, 321 Church St. SE, Minneapolis, MN 55455, USA

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Corresponding author: E-mail address: zbcui@ihb.ac.cn (Zongbin Cui)
Received Date: 2012-05-03
Accepted Date: 2012-05-28
Rev Recd Date: 2012-05-21

Available Online: 2012-08-23

Publish Date: 2012-09-20

Abstract

Abstract

Expression-independent gene or polyadenylation [poly(A)] trapping is a powerful tool for genome-wide mutagenesis regardless of whether a targeted gene is expressed. Although a number of poly(A)-trap vectors have been developed for the capture and mutation of genes across a vertebrate genome, further efforts are needed to avoid the 3′-terminal insertion bias and the splice donor (SD) read-through, and to improve the mutagenicity. Here, we present aSleeping Beauty (SB) transposon-based vector that can overcome these limitations through the inclusion of three functional cassettes required for gene-finding, gene-breaking and large-scale mutagenesis, respectively. The functional cassette contained a reporter/selective marker gene driven by a constitutive promoter in front of a strong SD signal and an AU-rich RNA-destabilizing element (ARE), which greatly reduced the SD read-through events, except that the internal ribosomal entry site (IRES) element was introduced in front of the SD signal to overcome the phenomenon of 3′-bias gene trapping. The breaking cassette consisting of an enhanced splicing acceptor (SA), a poly(A) signal coupled with a transcriptional terminator (TT) effectively disrupted the transcription of trapped genes. Moreover, theHsp70 promoter from tilapia genome was employed to drive the inducible expression of SB11, which allows the conditional remobilization of a trap insert from a non-coding region. The combination of three cassettes led to effective capture and disruption of endogenous genes in HeLa cells. In addition, the Cre/LoxP system was introduced to delete the Hsp70-SB11 cassette for stabilization of trapped gene interruption and biosafety. Thus, this poly(A)-trap vector is an alternative and effective tool for identification and mutation of endogenous genes in cells and animals.
- Poly(A) trapping,
- Insertional mutagenesis,
- HeLa cells,
- Zebrafish embryos

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References(63)

References

[1]	Baker, K.E., Parker, R. Nonsense-mediated mRNA decay: terminating erroneous gene expression Curr. Opin. Cell Biol., 16 (2004),pp. 293-299
[2]	Chen, W.V., Delrow, J., Corrin, P.D. et al. Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis Nat. Genet., 36 (2004),pp. 304-312
[3]	Clark, K.J., Balciunas, D., Pogoda, H.M. et al. Nat. Methods, 8 (2011),pp. 506-515
[4]	Clark, K.J., Geurts, A.M., Bell, J.B. et al. Transposon vectors for gene-trap insertional mutagenesis in vertebrates Genesis, 39 (2004),pp. 225-233
[5]	Cui, Z., Geurts, A.M., Liu, G. et al. J. Mol. Biol., 318 (2002),pp. 1221-1235
[6]	De-Zolt, S., Altschmied, J., Ruiz, P. et al. Gene-trap vectors and mutagenesis Methods Mol. Biol., 530 (2009),pp. 29-47
[7]	Ellingsen, S., Laplante, M.A., Konig, M. et al. Large-scale enhancer detection in the zebrafish genome Development, 132 (2005),pp. 3799-3811
[8]	Ellis, J. Silencing and variegation of gamma retrovirus and lentivirus vectors Hum. Gene Ther., 16 (2005),pp. 1241-1246
[9]	Galla, M., Schambach, A., Falk, C.S. et al. Avoiding cytotoxicity of transposases by dose-controlled mRNA delivery Nucleic Acids Res., 39 (2011),pp. 7147-7160
[10]	Geurts, A.M., Wilber, A., Carlson, C.M. et al. BMC Biotechnol., 6 (2006),p. 30
[11]	Geurts, A.M., Yang, Y., Clark, K.J. et al. Mol. Ther., 8 (2003),pp. 108-117
[12]	Gossler, A., Joyner, A.L., Rossant, J. et al. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes Science, 244 (1989),pp. 463-465
[13]	Grabundzija, I., Irgang, M., Mates, L. et al. Comparative analysis of transposable element vector systems in human cells Mol. Ther., 18 (2010),pp. 1200-1209
[14]	Guan, C., Ye, C., Yang, X. et al. A review of current large-scale mouse knockout efforts Genesis, 48 (2010),pp. 73-85
[15]	Hackett, P.B., Ekker, S.C., Largaespada, D.A. et al. Adv. Genet., 54 (2005),pp. 189-232
[16]	Hansen, J., Floss, T., Van Sloun, P. et al. A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome Proc. Natl. Acad. Sci. USA, 100 (2003),pp. 9918-9922
[17]	Hirashima, M., Bernstein, A., Stanford, W.L. et al. Gene-trap expression screening to identify endothelial-specific genes Blood, 104 (2004),pp. 711-718
[18]	Huang, X., Guo, H., Tammana, S. et al. Mol. Ther., 18 (2010),pp. 1803-1813
[19]	Ishida, Y., Leder, P. RET: a poly A-trap retrovirus vector for reversible disruption and expression monitoring of genes in living cells Nucleic Acids Res., 27 (1999),p. e35
[20]	Ivics, Z., Hackett, P.B., Plasterk, R.H. et al. Cell, 91 (1997),pp. 501-510
[21]	Ivics, Z., Izsvak, Z. The expanding universe of transposon technologies for gene and cell engineering Mob. DNA, 1 (2010),p. 25
[22]	Ivics, Z., Li, M.A., Mates, L. et al. Transposon-mediated genome manipulation in vertebrates Nat. Methods, 6 (2009),pp. 415-422
[23]	Izsvak, Z., Ivics, Z. Nat. Methods, 2 (2005),pp. 735-736
[24]	Jenkins, N.A., Dupuy, A.J., Akagi, K. et al. Nature, 436 (2005),pp. 221-226
[25]	Kawakami, K., Takeda, H., Kawakami, N. et al. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish Dev. Cell, 7 (2004),pp. 133-144
[26]	Keng, V.W., Villanueva, A., Chiang, D.Y. et al. A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma Nat. Biotechnol., 27 (2009),pp. 264-274
[27]	Kong, J., Wang, F., Brenton, J.D. et al. Nucleic Acids Res., 38 (2010),p. e173
[28]	Largaespada, D.A., Collier, L.S., Carlson, C.M. et al. Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse Nature, 436 (2005),pp. 272-276
[29]	Liang, Q., Kong, J., Stalker, J. et al. Genesis, 47 (2009),pp. 404-408
[30]	Lin, Q., Donahue, S.L., Moore-Jarrett, T. et al. Mutagenesis of diploid mammalian genes by gene entrapment Nucleic Acids Res., 34 (2006),p. e139
[31]	Liu, G., Aronovich, E.L., Cui, Z. et al. J. Gene Med., 6 (2004),pp. 574-583
[32]	Liu, G., Geurts, A.M., Yae, K. et al. J. Mol. Biol., 346 (2005),pp. 161-173
[33]	Liu, Z.J., Zhu, Z.Y., Roberg, K. et al. DNA Seq., 1 (1990),pp. 125-136
[34]	Livak, K.J., Schmittgen, T.D. Methods, 25 (2001),pp. 402-408
[35]	Matsuda, E., Shigeoka, T., Iida, R. et al. Proc. Natl. Acad. Sci. USA, 101 (2004),pp. 4170-4174
[36]	Medico, E., Gambarotta, G., Gentile, A. et al. A gene trap vector system for identifying transcriptionally responsive genes Nat. Biotechnol., 19 (2001),pp. 579-582
[37]	Molina, A., Di Martino, E., Martial, J.A. et al. Heat shock stimulation of a tilapia heat shock protein 70 promoter is mediated by a distal element Biochem. J., 356 (2001),pp. 353-359
[38]	Nord, A.S., Vranizan, K., Tingley, W. et al. Modeling insertional mutagenesis using gene length and expression in murine embryonic stem cells PLoS ONE, 2 (2007),p. e617
[39]	Plasterk, R.H., Izsvak, Z., Ivics, Z. Resident aliens: the Tc1/mariner superfamily of transposable elements Trends Genet., 15 (1999),pp. 326-332
[40]	Rad, R., Rad, L., Wang, W. et al. Science, 330 (2010),pp. 1104-1107
[41]	Ruley, H.E., Osipovich, A.B., Singh, A. Post-entrapment genome engineering: first exon size does not affect the expression of fusion transcripts generated by gene entrapment Genome Res., 15 (2005),pp. 428-435
[42]	Sato, K., Ito, R., Baek, K.H. et al. A specific DNA sequence controls termination of transcription in the gastrin gene Mol. Cell. Biol., 6 (1986),pp. 1032-1043
[43]	Schnutgen, F., Hansen, J., De-Zolt, S. et al. Enhanced gene trapping in mouse embryonic stem cells Nucleic Acids Res., 36 (2008),p. e133
[44]	Shigeoka, T., Kawaichi, M., Ishida, Y. Suppression of nonsense-mediated mRNA decay permits unbiased gene trapping in mouse embryonic stem cells Nucleic Acids Res., 33 (2005),p. e20
[45]	Sinzelle, L., Kapitonov, V.V., Grzela, D.P. et al. Transposition of a reconstructed Harbinger element in human cells and functional homology with two transposon-derived cellular genes Proc. Natl. Acad. Sci. USA, 105 (2008),pp. 4715-4720
[46]	Sivasubbu, S., Balciunas, D., Davidson, A.E. et al. Gene-breaking transposon mutagenesis reveals an essential role for histone H2afza in zebrafish larval development Mech. Dev., 123 (2006),pp. 513-529
[47]	Skarnes, W.C. Two ways to trap a gene in mice Proc. Natl. Acad. Sci. USA, 102 (2005),pp. 13001-13002
[48]	Skarnes, W.C., von Melchner, H., Wurst, W. et al. A public gene trap resource for mouse functional genomics Nat. Genet., 36 (2004),pp. 543-544
[49]	Stanford, W.L., Cohn, J.B., Cordes, S.P. Gene-trap mutagenesis: past, present and beyond Nat. Rev. Genet., 2 (2001),pp. 756-768
[50]	Takeda, J., Keng, V.W., Yae, K. et al. Nat. Methods, 2 (2005),pp. 763-769
[51]	Tsakiridis, A., Tzouanacou, E., Rahman, A. et al. Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells Nucleic Acids Res., 37 (2009),p. e129
[52]	Urasaki, A., Asakawa, K., Kawakami, K. Proc. Natl. Acad. Sci. USA, 105 (2008),pp. 19827-19832
[53]	Uren, A.G., Kool, J., Berns, A. et al. Retroviral insertional mutagenesis: past, present and future Oncogene, 24 (2005),pp. 7656-7672
[54]	Uren, A.G., Mikkers, H., Kool, J. et al. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites Nat. Protoc., 4 (2009),pp. 789-798
[55]	von Melchner, H., Schnutgen, F., De-Zolt, S. et al. Genomewide production of multipurpose alleles for the functional analysis of the mouse genome Proc. Natl. Acad. Sci. USA, 102 (2005),pp. 7221-7226
[56]	Wang, X.Q., Luk, J.M., Leung, P.P. et al. Alternative mRNA splicing of liver intestine-cadherin in hepatocellular carcinoma Clin. Cancer Res., 11 (2005),pp. 483-489
[57]	Wilson, M.H., Coates, C.J., Mol. Ther., 15 (2007),pp. 139-145
[58]	Xu, N., Chen, C.Y., Shyu, A.B. Modulation of the fate of cytoplasmic mRNA by AU-rich elements: key sequence features controlling mRNA deadenylation and decay Mol. Cell. Biol., 17 (1997),pp. 4611-4621
[59]	Yant, S.R., Kay, M.A. Mol. Cell. Biol., 23 (2003),pp. 8505-8518
[60]	Yant, S.R., Wu, X., Huang, Y. et al. High-resolution genome-wide mapping of transposon integration in mammals Mol. Cell. Biol., 25 (2005),pp. 2085-2094
[61]	Yergeau, D.A., Kelley, C.M., Kuliyev, E. et al. BMC Dev. Biol., 10 (2010),p. 11
[62]	Zambrowicz, B.P., Abuin, A., Ramirez-Solis, R. et al. Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention Proc. Natl. Acad. Sci. USA, 100 (2003),pp. 14109-14114
[63]	Zayed, H., Izsvak, Z., Walisko, O. et al. Mol. Ther., 9 (2004),pp. 292-304