A highly efficient recombineering-based method for generating conditional knockout mutations

Abstract

Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. This method uses homologous recombination mediated by the lambda phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce loxP or FRT sites into the subcloned DNA. Unlike other methods that use short 45-55-bp regions of homology for recombineering, our method uses much longer regions of homology. We also make use of several new E. coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive lambda prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter. We also describe two new Neo selection cassettes that work well in both E. coli and mouse ES cells. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk. This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes.

Figure 1.

Subcloning a DNA fragment from a BAC into pBluescript (pSK⁺) by gap repair with short homology arms via recombineering. Primers that have 20 bp of homology (yellow arrows) to pBluescript (yellow circle) at their 3′ end, and 50 bp (purple or blue) of homology at their 5′ ends to one of two ends of the BAC DNA to be subcloned (light blue), are used to amplify pBluescript. The PCR-amplified, linearized pBluescript containing the two homology arms is then transformed into recombination-competent cells that carry the BAC (BAC backbone in pink color). Gap-repaired plasmids are selected by their ampicillin resistance. The black bar denotes the location of Evi9 exon 4.

Figure 2.

An improved procedure for subcloning DNA from BACs and for constructing cko-targeting vectors. The homology arms used for gap repair (subcloning) and for targeting, are PCR-amplified from BAC DNA. The two homology arms (purple or dark blue), amplified using primers A and B or primers Y and Z, were cloned into an MC1TK-containing plasmid, to generate the gap repair (retrieval) plasmid for subcloning. The gap repair plasmid was linearized with HindIII to create a DNA double-strand break for gap repair. A minitargeting vector was constructed by ligating together the two PCR products generated by amplification of BAC DNA with primers C and D (light green) or primers E and F (blue), a floxed Neo selection cassette (black arrow: loxP site), and pBluescript. A BglII restriction site was included in the minitargeting vector for diagnosing gene targeting in ES cells. The black arrows denote loxP sites. The targeting cassette was excised by NotI and SalI digestion, or by PCR amplification, using primers C and F. The gap-repaired plasmid and the excised targeting cassette were cotransformed into recombination-competent DY380 or EL350 cells. The recombinants had a floxed Neo cassette inserted between primers D and E and can be selected on kanamycin plates. The Neo cassette was excised with Cre recombinase, leaving a single loxP site at the targeted locus (see Fig. 3). Similarly, a Neo selection cassette can be inserted between primers H and I using homology arms amplified by primers G, H (light orange), and I, J (light purple).

Figure 3.

Constructing an Evi9 conditional knockout allele. (A) The 11.0-kb genomic DNA fragment containing Evi9 exon 4 was subcloned from BAC-A12 using gap repair. EcoRV digestion of the gap-repaired plasmid generates 7.6-kb and 8.8-kb fragments. The 7.6-kb fragment contains Evi9 exon 4 sequences, whereas the 8.8-kb fragment, common to all lanes, contains plasmid sequences and Evi9 sequences located upstream of exon 4. The floxedNeo cassette of PL452 was targeted upstream of Evi9 exon 4. In the targeted plasmid, the 7.6-kb EcoRV fragment increases in size to 9.6 kb because of the addition of the floxedNeo cassette. Excision of the floxed Neo cassette leaves behind a single loxP (black arrow) at the targeted locus, and the normal EcoRV digestion pattern is restored. Next, the PL451 selection cassette, containing the Neo gene flanked by FRT sites (green arrow) and a downstream loxP, was targeted downstream of Evi9 exon 4. The PL451 selection cassette contains an EcoRV site, which results in the production of 6.5-kb and 3.1-kb fragments following EcoRV digestion. This is the Evi9 cko-targeting vector. To test the functionality of the FRT sites in the cko-targeting vector, the PL451 selection cassette was excised from the cko-targeting vector by FLP recombinase following electroporation into EL250 cells. This reduces the size of the 6.5-kb EcoRV fragment to 4.5 kb. Finally, electroporation of the cko-targeting cassette into EL350 cells expressing Cre recombinase excises the entire DNA between the two loxP sites, creating a 4.6-kb EcoRV fragment. (B) EcoRV-digestion patterns of the plasmids at every stage of the targeting vector construction.

Figure 4.

Identification of correctly targeted ES cell clones. (A) Homologous recombination between the Evi9 cko-targeting vector and the Evi9 genomic locus. Correctly targeted ES cells (cko allele) have a 5.5-kb BglII band, in addition to an 18.1-kb wild-type band, following hybridization with the 5′ probe. These cko clones also have a 6.3-kb EcoRV-targeted band, as well as a 7.3-kb wild-type band, following hybridization with the 3′ probe. (B) Southern blot analysis of the ES cell clones. The 5′ probe was used in the left panel, and a 3′ probe was used in the right panel. (wt) Wild-type ES clones; (cko) conditional knockout ES clones.

Figure 5.

Flow chart showing the different steps used in making a conditional knockout targeting vector by recombineering.