Figure 1

A simple and efficient method to rapidly construct exon-trapping targeting vectors. a Schematic representation of entry clones with floxed promoterless markers. For simplicity, the plasmid backbone is not drawn. IRES internal ribosome entry site, 2A a 2A-peptide sequence derived from Thosea asigna virus (TaV), Puro ^R puromycin-resistance gene, Hyg ^R hygromycin-resistance gene, Neo ^R neomycin-resistance gene, βgeo lacZ/Neo ^R, EGFP enhanced green fluorescent protein gene, pA polyadenylation signal. Half-closed triangles and closed triangles represent lox71 and loxP sequences, respectively. b Primer design for PCR amplification of homology arms. Each primer has four guanine residues at the 5′ end followed by an attB sequence. The four attB sequences attB4, attB1, attB2 and attB3 differ from one another, enabling efficient site-specific BP and LR recombination. The 5′-arm reverse primer should be set on the exon to be trapped (i.e., exon X in panel c), in order for the 5′-arm fragment to possess an authentic splice acceptor site at the 3′ side. The I-SceI site added to the 3′-arm reverse primer facilitates linearization of the resulting targeting vector. GSS gene-specific sequences. See text for details. c Flow diagram of construction of targeting vectors based on the MultiSite Gateway system, which consists of three steps: (1) PCR amplification with attB-containing primers, (2) BP recombination between 5′ or 3′ arm fragment and a donor vector (pDONR P4-P1R or pDONR P2R-P3, respectively), and (3) LR recombination to yield the targeting vector by one-time assembly of four DNA fragments (see text for details). SA splice acceptor site, drug ^R drug-resistance gene, Km ^R kanamycin-resistance gene, Amp ^R ampicillin-resistance gene. d Schematic representation of pENTR SA-IRES-Puro and pENTR SA-IRES-Hyg. These two entry clones harbor an SA site-linked promoterless marker gene. See “Methods” for details.