Novel strategies for gene trapping and insertional mutagenesis mediated by Sleeping Beauty transposon

Abstract

Gene and poly(A) trappings are high-throughput approaches to capture and interrupt the expression of endogenous genes within a target genome. Although a number of trapping vectors have been developed for investigation of gene functions in cells and vertebrate models, there is still room for the improvement of their efficiency and sensitivity. Recently, two novel trapping vectors mediated by Sleeping Beauty (SB) transposon have been generated by the combination of three functional cassettes that are required for finding endogenous genes, disrupting the expression of trapped genes, and inducing the excision of integrated traps from their original insertion sites and then inserting into another gene. In addition, several other strategies are utilized to improve the activities of two trapping vectors. First, activities of all components were examined in vitro before the generation of two vectors. Second, the inducible promoter from the tilapia Hsp70 gene was used to drive the expression of SB gene, which can mediate the excision of integrated transposons upon induction at 37 °C. Third, the Cre/LoxP system was introduced to delete the SB expression cassette for stabilization of gene interruption and bio-safety. Fourth, three stop codons in different reading frames were introduced downstream of a strong splice acceptor (SA) in the gene trapping vector to effectively terminate the translation of trapped endogenous genes. Fifth, the strong splicing donor (SD) and AU-rich RNA-destabilizing element exhibited no obvious insertion bias and markedly reduced SD read-through events, and the combination of an enhanced SA, a poly(A) signal and a transcript terminator in the poly(A) trapping vector efficiently disrupted the transcription of trapped genes. Thus, these two trapping vectors are alternative and effective tools for large-scale identification and disruption of endogenous genes in vertebrate cells and animals.

Keywords: Sleeping Beauty; gene trapping; insertional mutagenesis; poly(A) trapping; transposon.

Figure 1. The flowchart of gene trapping with pT2/Gene-Trap. Insertion of the gene-Trap cassette into an intron of transcriptionally active loci can generate a fusion transcript that contains the upstream exon and the reporter marker. The expression of SB11 gene was driven by the heat-inducible promoter TiHsp70. IR/DR(L) and IR/DR(R), left and right inverted repeat/directed repeat of the SB transposon; SA, splice acceptor; IRES, internal ribosome entry site; GFP, GFP reporter gene; poly(A), poly(A) signal; TiHsp70, tilapia Hsp70 promoter; SB11, SB11 transposase gene.

Figure 2. The flowchart of gene trapping with pT2/Poly(A)-Trap. Insertion of the poly(A)-trap cassette into an intron before a functional poly(A) sequence for an endogenous gene leads to the generation of a stable pre-mRNA and the proper splicing between the trap SD with a SA downstream of the insertion site and gives rise to a fusion transcript for GFP expression. The proper expression of trapped gene was disrupted by the formation of another fusion transcript that contains exons upstream of the insert site. The expression of SB11 gene was driven by the heat-inducible promoter TiHsp70. IR/DR(L) and IR/DR(R), left and right inverted/directed repeats of the SB transposon; SA, splice acceptor from carp β-actin gene; Exon, the exon 2 from the carp β-actin gene; SV40 poly(A), SV40 polyadenylation sequence; TT, transcript terminator sequence. TiHSP70, tilapia Hsp70 promotor; SB11, SB11 transposase gene. polyA, a poly(A) signal from carp β-actin gene; SV40, SV40 promoter; GFP, GFP reporter gene; IRES, internal ribosome entry site; SD, splice donor; ARE, AU-riched element.

Figure 3. The schematic of gene trapping in zebrafish. The transposon-based gene trapping constructs pT2/Gene-Trap or pT2/Poly(A)-Trap and in vitro synthesized SB11 mRNA were co-microinjected into one-cell stage zebrafish embryos to generate transgenic zebrafish lines carrying the gene trapping cassette. Transgenic individuals expressing GFP can be directly used for the analysis of interrupted genes, while other adults showing no GFP expression can be treated with heat-shock during spermatogenesis or oogenesis to obtain offspring with novel insertions and GFP expression. A few of zebrafish individuals expressing GFP in distinct tissues are shown in the right panel.