Abstract
Gene trapping is a high-throughput approach to elucidate gene functions by disrupting and recapitulating expression of genes in a target genome. A number of transposon-based gene-trapping systems are developed for mutagenesis in cells and model organisms, but there is still much room for the improvement of their efficiency in gene disruption and mutation. Herein, a gene-trapping system mediated by Sleeping Beauty (SB) transposon was developed by inclusion of three functional cassettes. The mutation cassette can abrogate the splice of trapped genes and terminate their translation. Once an endogenous gene is captured, the finding cassette independently drives the translation of reporter gene in HeLa cells and zebrafish embryos. The efficiency cassette controls the remobilization of integrated traps through inducible expression of SB gene. Analysis of transposon-genome junctions indicate that most of trap cassettes are integrated into an intron without an obvious 3′ bias. The transcription of trapped genes was abrogated by alternative splicing of the mutation cassette. In addition, integrated transposons can be induced to excise from their original insertion sites. Furthermore, the Cre/LoxP system was introduced to delete the efficiency cassette for stabilization of gene interruption and bio-safety. Thus, this gene-trap vector is an alternative and effective tool for the capture and disruption of endogenous genes in vitro and in vivo.
Highlights
The completion of genome projects for human and other model species has advanced biological researches into the post-genome era
Generation of a Novel Gene-trap Vector there are several versions of transposon-based gene trapping vectors that are used in various vertebrate systems [53,54,55,56], there is room for improvement to increase gene tag and mutation efficiency
This strong splice acceptor signal combined with the polyadenylation signal and transcriptional termination element in our gene-trap vector disrupted the transcription of trapped endogenous genes efficiently
Summary
The completion of genome projects for human and other model species has advanced biological researches into the post-genome era. Mutagenesis approaches including Nethyl-N-nitrosourea (ENU)-induced mutations [1,2], Cre/loxPmediated gene targeting [3], retrovirus- and transposon-based gene trapping [4,5,6], are extensively developed to disrupt expression of genes in model organisms including mouse and zebrafish. Limitations of ENU and other chemical mutagenesis approaches remain the identification of genes whose mutations are responsible for a particular phenotype [9,10] and the laborious and frustrating tasks of positional cloning. The development of gene targeting is based on homologous recombination and the availability of embryonic stem cells, and this technique is widely used for the generation of knock-out mouse [11,12]. Since the complete deletion of some genes by conventional gene targeting could be lethal to embryonic development [13,14], conditional knock-out techinques are alternatively developed [15,16]; the procedures of these approaches are extremely laborious and time consuming
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