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

Song, Guangliang; Long, Yong; Hackett, Perry B.

doi:10.1016/j.jgg.2012.05.010

Cited by 14 publications

(16 citation statements)

References 64 publications

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“…To circumvent this problem, the Cre/loxP system (Van Duyne 2001) was introduced into the SB transposon to conditionally delete the SB11 gene for stabilization of disrupted genes. These elements have been found to work very well in zebrafish in our previous studies (Song et al 2012a;Song et al 2012b). Thus, the application of this SB transposon-mediated transgenic system appears to bring significant improvements with regard to efficiency, precision and safety of gene transfer.…”

Section: Discussionmentioning

confidence: 68%

Gene transfer and mutagenesis mediated by Sleeping Beauty transposon in Nile tilapia (Oreochromis niloticus)

Long

et al. 2013

Transgenic Res

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The success of gene transfer has been demonstrated in many of vertebrate species, whereas the efficiency of producing transgenic animals remains pretty low due to the random integration of foreign genes into a recipient genome. The Sleeping Beauty (SB) transposon is able to improve the efficiency of gene transfer in zebrafish and mouse, but its activity in tilapia (Oreochromis niloticus) has yet to be characterized. Herein, we demonstrate the potential of using the SB transposon system as an effective tool for gene transfer and insertional mutagenesis in tilapia. A transgenic construct pT2/tiHsp70-SB11 was generated by subcloning the promoter of tilapia heat shock protein 70 (tiHsp70) gene, the SB11 transposase gene and the carp b-actin gene polyadenylation signal into the second generation of SB transposon. Transgenic tilapia was produced by microinjection of this construct with in vitro synthesized capped SB11 mRNA. SB11 transposon was detected in 28.89 % of founders, 12.9 % of F1 and 43.75 % of F2. Analysis of genomic sequences flanking integrated transposons indicates that this transgenic tilapia line carries two copies of SB transposon, which landed into two different endogenous genes. Induced expression of SB11 gene after heat shock was detected using reverse transcription PCR in F2 transgenic individuals. In addition, the Cre/loxP system was introduced to delete the SB11 cassette for stabilization of gene interruption and biosafety. These findings suggest that the SB transposon system is active and can be used for efficient gene transfer and insertional mutagenesis in tilapia.

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Section: Discussionmentioning

confidence: 68%

Gene transfer and mutagenesis mediated by Sleeping Beauty transposon in Nile tilapia (Oreochromis niloticus)

Long

et al. 2013

Transgenic Res

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“…Although the polyA trap design should trap all genes regardless of expression levels, the results of polyA trapping screens have often been below expectations (Zambrowicz et al, 2003;Shigeoka et al, 2005). To overcome this limitation, efforts have been made to combine different promoters and splice donor (SD) signals, insert a synthetic intron within the selectable marker gene, or include the Sleeping Beauty transposon (Lin et al, 2006;Tsakiridis et al, 2009;Song et al, 2012). However, compared with the promoter-trapping strategy, polyA trapping has not been validated in a large-scale gene trap setting.…”

Section: Discussionmentioning

confidence: 99%

A promoter trap vector for knocking out bovine myostatin gene with high targeting efficiency

Zhao

Liang

et al. 2015

Genet. Mol. Res.

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ABSTRACT. With the development of gene targeting approaches, genomic mutation technologies in livestock animals such as gene trapping, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats and their associated systems have been improved. Although ZFNs have been used for gene targeting in many species, the off-target sites are still present. Using gene trapping, the workload of screening of targeted clones was decreased by generating a smaller number of drugresistant clones. Determining whether the efficiency of gene trapping is lower than that of ZFNs for a specific gene has been challenging. In this study, to knock out the bovine myostatin gene, we constructed a promoter trap vector and compared its efficiency with that of ZFNs. The promoter trap vector contained a green fluorescent protein sequence without the promoter and a neomycin phosphotransferase (neo R ) cassette driven by the phosphoglycerate kinase promoter. When the trapping vector was inserted downstream of the endogenous promoter, the fluorescent protein gene was expressed. The targeted-positive cell clones were identified based on green fluorescence and G418 double selection, followed by polymerase chain reaction analysis and sequencing. The targeting efficiency reached 5%. Compared with the efficiency of ZFN pairs (5.17 and 2.86%), the promoter trap vector PIII-myostatin could knock out the bovine myostatin gene. Therefore, gene trapping may be an effective tool for genomic modification.

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“…The greatest success of SB has been in its use for phenotype-driven genetics (Carlson et al, 2003;Elso et al, 2015;Horie et al, 2003;Ivics & Izsvak, 2011;Izsvak et al, 2010;Moriarity & Largaespada, 2011). Unlike a candidate-based strategy, SB transposon-based gene trapping, enhancer trapping and unbiased mutagenesis approach is suitable to identify genes with unexpected phenotypes (Balciunas et al, 2004;Lu et al, 2007;Song & Cui, 2013).…”

Section: Awakening Sleeping Beauty In the Clinicmentioning

confidence: 99%

Sleeping Beautytransposition: from biology to applications

Narayanavari

Chilkunda

Ivics

et al. 2016

Critical Reviews in Biochemistry and Molecular Biology

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Sleeping Beauty (SB) is the first synthetic DNA transposon that was shown to be active in a wide variety of species. Here, we review studies from the last two decades addressing both basic biology and applications of this transposon. We discuss how host-transposon interaction modulates transposition at different steps of the transposition reaction. We also discuss how the transposon was translated for gene delivery and gene discovery purposes. We critically review the system in clinical, pre-clinical and non-clinical settings as a non-viral gene delivery tool in comparison with viral technologies. We also discuss emerging SB-based hybrid vectors aimed at combining the attractive safety features of the transposon with effective viral delivery. The success of the SBbased technology can be fundamentally attributed to being able to insert fairly randomly into genomic regions that allow stable long-term expression of the delivered transgene cassette. SB has emerged as an efficient and economical toolkit for safe and efficient gene delivery for medical applications.ARTICLE HISTORY

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

Cited by 14 publications

References 64 publications

Gene transfer and mutagenesis mediated by Sleeping Beauty transposon in Nile tilapia (Oreochromis niloticus)

Gene transfer and mutagenesis mediated by Sleeping Beauty transposon in Nile tilapia (Oreochromis niloticus)

A promoter trap vector for knocking out bovine myostatin gene with high targeting efficiency

Sleeping Beautytransposition: from biology to applications

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