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

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)

摘要

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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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参考文献(63)

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