Recombinase-mediated cassette exchange provides a versatile platform for gene targeting: knockout of miR-31b

Abstract

A series of vectors has been designed to enhance the versatility of targeted homologous recombination. Recombinase-mediated cassette exchange permits sequential targeting at any locus and improves flexibility in making user-defined mutations. Application of RMCE to delete an intronic microRNA gene is described.

Figure 1.—

Schematic representation of ends-out gene targeting by homologous recombination. Three- to 4-kb homology sequences flanking the target gene are cloned into the pW25 ends-out vector. P-element-mediated transformation gives rise to transgenic donor containing the targeting cassette. Induction of FLP recombinase is used to excise a circular DNA molecule containing the targeting vector, which is then linearized by cleavage with the I-SceI meganuclease. The linearized targeting vector can then recombine with the chromosomal target locus, replacing the endogenous gene with mini-white and generating a mutant heterozygous for the targeted gene.

Figure 2.—

Modified ends-out gene targeting vectors. (A) pW25 vector. Arrowheads are P-element ends, half-arrows are FRT sequences, hatched boxes are I-SceI site, open boxes are cloning sites for homologous DNA sequences, diamonds are six-frame translation stop codons, and black boxes are the mini-white marker gene. (B) pW25-Gal4 vector. The Gal4 coding sequence, denoted by hatched pentagon, was cloned into the pW25 vector, between the 5′ six-frame stop codons and the loxP site. (C) pW25-RMCE vector. A 221-bp fragment for the phage attachment site (attP, denoted by black triangles,) was PCR amplified from the pTA-attP plasmid and inverted attP fragments were cloned into KpnI and AscI sites upstream and downstream of the mini-white sequence, respectively. (D and E) pW25-attB and pW25-Gal4-attB1 vectors. A 285-bp fragment containing the bacterial phage attachment site (attB, denoted by gray triangles) was cloned into both pW25 and pW25-Gal4 vector backbones at the NdeI site. (F) pW25-Gal4-attB2 vector. The Gal4 coding sequence was cloned between the 5′ loxP site and mini-white by composite cloning using pW25-attB as the vector backbone. This allows removal of the Gal4 driver together with the mini-white marker using the Cre-LoxP recombinase system. Restriction enzyme sites for PacI and FseI were introduced to the multiple cloning sites at the 5′ and 3′ end, respectively, to facilitate directional cloning of “homology arms.”

Figure 3.—

Site-specific integration via ϕC31 integrase-mediated RMCE. (A) Schematic representation of the targeting event replacing miR-31b with att-P and loxP flanked mini-white in the intron of CG10962. Exchange of the mini-white marker with GFP by crossover between the two inverted attP and attB sites leads to clean exchange of mini-white by GFP, resulting in a new attR site, denoted by checkered triangles. Cassette exchange can occur in both orientations. Primer pairs (arrows) were designed to distinguish the two outcomes. (B) Quantitative microRNA PCR to measure the level of miR-31b in total RNA from control male flies (w¹¹¹⁸), males carrying the mini-white targeted allele (miR-31-w+) and males carrying the GFP replacement alllele (illustrated in C).(C) PCR genotyping of an RMCE candidate with GFP in orientation 1. Lanes 1 and 2: failure to amplify with primer pairs p5/p6 and p7/p8 indicates the absence of GFP in orientation 2. Lanes 3 and 4: the product amplified primer pairs p1/p2 and p3/p4 indicates the presence of GFP in orientation 1. Lanes 5 and 6 show the specificity of primer pairs p1/p2 and p3/p4 using initial mini-white-containing mutant genomic DNA as the template. (D) Illustration of possible splicing patterns of CG10962 in various miR-31b mutants. Upper panel: CG10962-RA has 4 exons, denoted by gray boxes 1–4. miR-31b is present in the second intron. The mini-white marker in the targeting vector contains introns and exons of the white locus and could interfere with splicing of CG10962. Replacement of mini-white with an intronless GFP cassette is illustrated. Lower panel: Primers p9/p10 amplify a product spanning the exon 2 and exon 3 junction and can be used in RT–PCR to measure the efficiency of removal of intron 2. (E) Quantitative RT–PCR using primers p9/10 (in D) showing exon 2/3 splicing of CG10962. Assays performed on total RNA from flies with genotypes as in B.