A simple polymerase chain reaction-based method for the construction of recombinase-mediated cassette exchange donor vectors

Abstract

Here we describe a simple method for generating donor vectors suitable for targeted transgenesis via recombinase-mediated cassette exchange (RMCE) using the PhiC31 integrase. This PCR-based strategy employs small attB "tails" on the primers used to amplify a sequence of interest, permitting the rapid creation of transgenes for in vivo analysis.

Figure 1.—

Site-specific integration via RMCE in Drosophila. The target cassette, a mini-white gene (orange line) flanked by inverted attP sites and borne on a P element, was integrated at several locations in the genome via standard P-element-mediated transgenesis (Spradling and Rubin 1982). In this study, we used the target P[attP.w+.attP] (GenBank accession EU761203), a derivative of pUASTP2 (Bateman et al. 2006) in which the UAS and TATA sequences have been removed; in prior unpublished presentations we have referred to this target as “PUASTP2.1” (see http://www.pairing.org/RMCE for further details). Target positions were determined by inverse PCR as previously described (Bateman et al. 2006). Introduction of a donor vector carrying a sequence of interest (blue line) flanked by inverted attB sites allows exchange of the cassettes in the presence of integrase, resulting in integration of the donor cassette. Shaded boxes, P-element ends; open triangles, att sites. Features are not drawn to scale.

Figure 2.—

RMCE using a genomic source of the ΦC31 integrase. The integrase is supplied via a nanos-integrase-NLS fusion, marked with a yellow⁺ transgene, that is carried on the X chromosome (Bischof et al. 2007), while the target cassette is carried on chromosome II. In the G₀ generation, males are backcrossed singly to two or three virgin females carrying the CyO balancer and dominant markers on the second chromosome, and their progeny are screened for candidate insertion events wherein the chromosome carrying the RMCE cassette has lost the mini-white gene.

Figure 3.—

AttB40 tails can be used to generate donor vectors via PCR. (A) Scheme for generating a donor vector via PCR. The attB40 sequence 5′-CGGGTGCCAGGGCGTGCCCTTGGGCTCCCCGGGCGCGTAC-3′ was added to the 5′-ends of the primers hsp70GFP5 (5′-CCGCTTTGGTACCTTTGCTA-3′) and hsp70GFP3 (5′-GCAGGTCGACCAATTCGAG-3′), which were subsequently used to amplify the GFP gene from the plasmid RINheXho-GFP (Wittkopp et al. 2002). The resulting fragment was gel purified using a kit from Qiagen and subcloned using Invitrogen's TOPO-TA cloning kit to create the donor vector pTA-attB40GFP. (B) Ethidium-bromide-stained gel of PCR products that were generated using primers with (right lane) and without (left lane) attB40 tails.