Effective gene trapping mediated by Sleeping Beauty transposon

PLoS One. 2012;7(8):e44123. doi: 10.1371/journal.pone.0044123. Epub 2012 Aug 31.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Base Sequence
  • Chromosomes, Human / genetics
  • DNA Transposable Elements / genetics*
  • Exons / genetics
  • Genes, Reporter
  • Genetic Techniques*
  • Genetic Vectors / genetics
  • Genome, Human / genetics
  • HSP70 Heat-Shock Proteins / genetics
  • HeLa Cells
  • Humans
  • Introns / genetics
  • Molecular Sequence Data
  • Mutagenesis, Insertional / genetics
  • Mutation / genetics
  • Promoter Regions, Genetic / genetics
  • Temperature
  • Tilapia / genetics
  • Transcription, Genetic
  • Zebrafish / embryology
  • Zebrafish / genetics

Substances

  • DNA Transposable Elements
  • HSP70 Heat-Shock Proteins

Grant support

This work was supported by the National Basic Research Program of China (#2012CB944500) and the National Natural Science Foundation of China (#30871442 to Z. Cui). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.