A hyperactive sleeping beauty transposase enhances transgenesis in zebrafish embryos
© Newman et al; licensee BioMed Central Ltd. 2010
Received: 21 June 2010
Accepted: 4 November 2010
Published: 4 November 2010
Transposons are useful molecular tools for transgenesis. The 'sleeping beauty' transposon is a synthetic member of the Tc1/mariner transposon family. Davidson et al. (2003) previously described a vector for zebrafish transgenesis consisting of the inverted repeats of 'sleeping beauty' flanking the gene to be transposed. Subsequently, there have been attempts to enhance the transpositional activity of 'sleeping beauty' by increasing the activity of its transposase. Recently, Mates et al. (2009) generated a hyperactive transposase giving a 100-fold increased transposition rate in mouse embryos.
The aim of this experiment was to determine whether this novel hyperactive transposase enhances transgenesis in zebrafish embryos. Using our previously characterised mitfa- amyloidβ-GFP transgene, we observed an eight-fold enhancement in transient transgenesis following detection of transgene expression in melanophores by whole mount in-situ hybridisation. However, high rates of defective embryogenesis were also observed.
The novel hyperactive 'sleeping beauty' transposase enhances the rate of transgenesis in zebrafish embryos.
Transposons direct integrations of single copies of genetic material into chromosomes  and are useful molecular tools for transgenesis in vertebrate species. They function by delivering a gene of interest to the chromosome in a cut and paste manner. The 'sleeping beauty' transposon is a synthetic member of the Tc1/mariner transposon family. The transposon was engineered from a consensus sequence of inactive fossil transposon sequences from various Salmonid fish genomes . Sleeping beauty consists of the transposase gene flanked by terminal inverted repeats of direct repeats. The transposase protein catalyses the excision and integration of donor DNA into a TA dinucleotide site of a recipient genome . The derived sleeping beauty vector system (SBT) has been shown to enhance production of transgeneic animals in comparison to simple methods of transgenesis such as injection of naked DNA [3, 4]. It is active in various vertebrate species such as fish, frogs, mice and rats [3, 5–7]. There have been attempts to enhance the transpositional activity of the SBT, specifically by increasing the activity of the transposase. Almost every amino acid has been changed to derive hyperactive mutants of the SB transposase and this has yielded modest increases in transpositional activity [8–11]. Recently, Mates et al. , used a large-scale genetic screen in mammalian cells to generate a hyperactive transposase that gave a ~100-fold enhancement of transpositional activity over the original SB transposase in mouse embryos.
Alzheimer's disease may be caused by the accumulation of amyloidβ peptides in the brain . Recently, we used the SBT system to generate a zebrafish melanophore model of amyloidβ toxicity . We generated transgenic zebrafish possessing human amyloidβ under the control of the mitfa promoter that drives expression specifically in melanophores (dark pigment cells) using our vector pT2-mitfa- amyloidβ-GFP. In that study the transposase mRNA was generated from the plasmid pSBRNAX that includes sequence from the 3' UTR of the Xenopus β-globin gene for mRNA stabilisation. In this experiment, we compared the rates of transient transgenesis in zebrafish embryos using the original transposase mRNA (SB10, generated from pSBRNAX ) or the hyperactive transposase mRNA (SB100, generated from pCMV(CAT)T7-SB100X ). It is important to note that the pCMV(CAT)T7-SB100X vector does not contain the Xenopus β-globin 3' UTR sequences for mRNA stabilisation. Therefore, SB100 mRNA may not be as stable as SB10 mRNA and, once injected into the zebrafish embryos, may possibly degrade at a faster rate.
Results of injections of transposase mRNAs alone or with the mitfa-amyloidβ-GFP transgene
Normal % (n)
Mild % (n)
Severe % (n)
mitfa- amyloidβ-GFP only
-with SB100 mRNA
-with SB10 mRNA
SB100 mRNA only
SB10 mRNA only
TOL2 mRNA only
In their tests of SB100-driven transgenesis in fertilised mouse oocytes, Mates et al. (2009) did not observe a decreased survival rate relative to uninjected controls at day 7 of mouse embryogenesis and high rates of transgenesis were observed in mouse litters. However, the slower rate of cell division that occurs in cleavage stage mouse embryos relative to zebrafish embryos may mean that the transposase mRNA breaks down in the mouse zygotes before it can cause developmental defects.
The enhancement of transgenesis in zebrafish embryos from use of the novel hyperactive transposase was not ~100-fold greater than the transgenesis rate using the original SB transposase. Nevertheless, the observed 8-fold increase is a considerable improvement for two reasons. First, the amyloidβ-GFP transgene is under the control of a tissue-specific promoter, mitfa, which directs expression of the transgene to melanophores. Melanophores make up only a small fraction of the total cells in a zebrafish embryo at 24 hpf. Thus, transient transgenesis is not expected to label this cell type frequently. Secondly, the SB10 mRNA is generated from pSBRNAX which has the Xenopus β-globin 3' UTR sequence for increased mRNA stability while the SB100 mRNA does not include such sequences. Therefore, the SB100 mRNA would be expected to degrade at a faster rate which might also affect transgenesis efficiency. If one considers that the germline transmission frequency of mitfa- amyloidβ-GFP in the original study using SB10 mRNA was 20% (for a <3% rate of observable transient transgenesis), then the 8-fold enhancement of transient transgenesis observed in this study would presumably further improve the rate of germline transgenesis in zebrafish. Overall, we conclude that the novel hyperactive 'sleeping beauty' transposase enhances the rate of transgenesis in zebrafish embryos.
Would like to thank Dr. Zsuzsanna Izsvak for providing the pCMV(CAT)T7-SB100X vector. This work was carried out under the auspices of the Animal Ethics Committee of The University of Adelaide. Research was supported by funds from the School of Molecular and Biomedical Research of The University of Adelaide, National Health and Medical research Council (NHMRC) Project Grant 453622 and the Cancer Council of South Australia.
- Wadman SA, Clark KJ, Hackett PB: Fishing for answers with transposons. Mar Biotechnol (NY). 2005, 7: 135-141. 10.1007/s10126-004-0068-2.View ArticleGoogle Scholar
- Ivics Z, Hackett PB, Plasterk RH, Izsvak Z: Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell. 1997, 91: 501-510. 10.1016/S0092-8674(00)80436-5.PubMedView ArticleGoogle Scholar
- Davidson AE, Balciunas D, Mohn D, Shaffer J, Hermanson S, Sivasubbu S, Cliff MP, Hackett PB, Ekker SC: Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon. Dev Biol. 2003, 263: 191-202. 10.1016/j.ydbio.2003.07.013.PubMedView ArticleGoogle Scholar
- Dupuy AJ, Clark K, Carlson CM, Fritz S, Davidson AE, Markley KM, Finley K, Fletcher CF, Ekker SC, Hackett PB, Horn S, Largaespada DA: Mammalian germ-line transgenesis by transposition. Proc Natl Acad Sci USA. 2002, 99: 4495-4499. 10.1073/pnas.062630599.PubMed CentralPubMedView ArticleGoogle Scholar
- Mates L, Chuah MK, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, Grzela DP, Schmitt A, Becker K, Matrai J, Ma L, Samara-Kuko E, Gysemans C, Pryputniewicz D, Miskey C, Fletcher B, Vandendriessche T, Ivics Z, Izsvak Z: Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009, 41: 753-761. 10.1038/ng.343.PubMedView ArticleGoogle Scholar
- Liu G, Aronovich EL, Cui Z, Whitley CB, Hackett PB: Excision of Sleeping Beauty transposons: parameters and applications to gene therapy. J Gene Med. 2004, 6: 574-583. 10.1002/jgm.486.PubMed CentralPubMedView ArticleGoogle Scholar
- Newman M, Wilson L, Camp E, Verdile G, Martins R, Lardelli M: A Zebrafish Melanophore Model of Amyloidbeta Toxicity. Zebrafish.
- Geurts AM, Yang Y, Clark KJ, Liu G, Cui Z, Dupuy AJ, Bell JB, Largaespada DA, Hackett PB: Gene transfer into genomes of human cells by the sleeping beauty transposon system. Mol Ther. 2003, 8: 108-117. 10.1016/S1525-0016(03)00099-6.PubMedView ArticleGoogle Scholar
- Yant SR, Park J, Huang Y, Mikkelsen JG, Kay MA: Mutational analysis of the N-terminal DNA-binding domain of sleeping beauty transposase: critical residues for DNA binding and hyperactivity in mammalian cells. Mol Cell Biol. 2004, 24: 9239-9247. 10.1128/MCB.24.20.9239-9247.2004.PubMed CentralPubMedView ArticleGoogle Scholar
- Zayed H, Izsvak Z, Walisko O, Ivics Z: Development of hyperactive sleeping beauty transposon vectors by mutational analysis. Mol Ther. 2004, 9: 292-304. 10.1016/j.ymthe.2003.11.024.PubMedView ArticleGoogle Scholar
- Baus J, Liu L, Heggestad AD, Sanz S, Fletcher BS: Hyperactive transposase mutants of the Sleeping Beauty transposon. Mol Ther. 2005, 12: 1148-1156. 10.1016/j.ymthe.2005.06.484.PubMedView ArticleGoogle Scholar
- Kirkitadze MD, Kowalska A: Molecular mechanisms initiating amyloid beta-fibril formation in Alzheimer's disease. Acta Biochim Pol. 2005, 52: 417-423.PubMedGoogle Scholar
- Jowett T: Tissue in situ hybridization. 1997, John Wiley and Sons, New YorkGoogle Scholar
- Higashijima S, Okamoto H, Ueno N, Hotta Y, Eguchi G: High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev Biol. 1997, 192: 289-299. 10.1006/dbio.1997.8779.PubMedView ArticleGoogle Scholar
- Kawakami K, Takeda H, Kawakami N, Kobayashi M, Matsuda N, Mishina M: A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev Cell. 2004, 7: 133-144. 10.1016/j.devcel.2004.06.005.PubMedView ArticleGoogle Scholar