Gene trap mutagenesis in the mouse

Abstract

Gene trapping in mouse embryonic stem (ES) cells is an efficient method for the mutagenesis of the mammalian genome. Insertion of a gene trap vector disrupts gene function, reports gene expression, and provides a convenient tag for the identification of the insertion site. The trap vector can be delivered to ES cells by electroporation of a plasmid, by retroviral infection, or by transposon-mediated insertion. Recent developments in trapping technology involve the utilization of site-specific recombination sites, which allow the induced modification of trap alleles in vitro and in vivo. Gene trapping strategies have also been successfully developed to screen for genes that are acting in specific biological pathways. In this chapter, we review different applications of gene trapping, and we provide detailed experimental protocols for gene trapping in ES cells by retroviral and transposon gene trap vectors.

Fig. 1

Schematic diagrams of different types of gene trap alleles A) Insertion of an enhancer trap vector with minimal promoter (mP) nearby a transcriptionally active gene activates expression of the β-galactosidase reporter (βgal). B) A promoter trap insertion, in which a splice acceptor (SA) fuses an endogenous transcript to a β-galactosidase/neomycin phophotransferase (βgeo) reporter/selection cassette. The fusion transcript of the promoter trap confers drug resistance, reports the expression pattern, and mutates the function of the endogenous trapped gene. Note that promoter trapping can also be performed with vectors that express drug resistance by a strong heterologous promoter (see text for details). C) The secretory trap vector is designed to trap genes that encode secretory signal peptide (SS). Secretion of the fusion protein from the endogenous gene and the trap vector is prevented by a transmembrane domain (TM) encoded in the trap vector. The TM anchors the fusion protein at the intracellular side of the cell membrane, thereby conferring drug resistance to trapped cells. D) The insertion of an exon trap vector into an exon of a transcriptionally active gene leads to a direct fusion of a selection gene (neomycin phosphotransferase, neo) to the endogenous gene. E) A polyA trap vector has two components: a upstream element with 5′ splice acceptor (SA βgal) to report gene expression, and a downstream element with promoter, selection gene, and splice donor (P-neo-SD), to select for drug-resistant intergenic insertions. F) The conditional “Flex” design contains a promoter trap cassette that is flanked by upstream and downstream arrays of heterologous recombination sites for the Flp (semicircles) and Cre recombinases (triangles); for details see (Schnütgen et al., 2005). Successive application of these recombinases leads to inversion (conditional allele) and re-inversion (re-instated mutagenic allele) of the promoter trap cassette. G) The conditional double switch vector contains an upstream 5′ splice acceptor-GFP reporter component (SA-IRESeGFP-pA), which is in inverse orientation to gene transcription and flanked by half mutant lox66 and lox71 sites (triangles). A downstream polyA trap component (P-puro-SD) is flanked by FRT recombination sites (semi-circles). Excision of the polyA trap component by Flp recombinase creates the conditional allele, and subsequent inversion of the reporter element generates the mutated allele (see (Xin et al., 2005) for details).

Fig. 2

Workflow of gene trapping by retroviral infection (A) or by electroporation with transposon vectors (B).

Fig. 3

Identification of gene trap insertion sites by Splinkerette PCR. (1) A DNA preparation of an ES cell clone is digested with BstYI restriction endonuclease. (2) ligation to the Splinkerette linker. (3) In the first round of PCR, only the vector-specific primer V1 can anneal and initiate synthesis of a strand that complements the Splinkerette linker. (4) The linker-specific primer L1 and the vector-specific primer V1 PCR amplify a junction fragment of the vector and the adjacent genomic DNA. (5) A PCR with nested primers L2 and V2 serves to improve specificity and yield of the reaction. (6) Generation of unspecific products is minimized, since the primer L1 cannot anneal to linkers that contains the self-complementary “hook” region.