Red recombination using PCR-amplified selectable markers is a well-established technique for mutagenesis of large DNA molecules in Escherichia coli. The system has limited efficacy and versatility, however, for markerless modifications including point mutations, deletions, and particularly insertions of longer sequences. Here we describe a procedure that combines Red recombination and cleavage with the homing endonuclease I-SceI to allow highly efficient, PCR-based DNA engineering without retention of unwanted foreign sequences. We applied the method to modification of bacterial artificial chromosome (BAC) constructs harboring an infectious herpesvirus clone to demonstrate the potential of the mutagenesis technique, which was used for the insertion of long sequences such as coding regions or promoters, introduction of point mutations, scarless deletions, and insertion of short sequences such as an epitope tag. The system proved to be highly reliable and efficient and can be adapted for a variety of different modifications of BAC clones, which are fundamental tools for applications as diverse as the generation of trans genic animals and the construction of gene therapy or vaccine vectors.
Bacterial artificial chromosomes (BACs), mini-F plasmids into which DNA of up to 300 kb can be cloned (1), have made possible rapid and efficient mutagenesis of large sequences by techniques that were previously restricted to genetic manipulation of small plasmids in Escherichia coli. This advance has accelerated progress in genome sequencing projects, has revolutionized work with DNA viruses (2,3), and has facilitated the generation of transgenic animals through the use of modified clones of genomic BAC libraries (4).
Besides RecA-mediated mutagenesis, Red recombination or RecET cloning is one of the most commonly exploited techniques to engineer sequences in large DNA constructs (5,6). Substrates for Red mutagenesis are DNA double-strand breaks (DSB) wherein homologous recombination is focused close to the ends of the linear double-stranded DNA (dsDNA). Because replication of the substrate is not required, Red recombination allows utilization of PCR-amplified positive selection markers using primers bearing 40–50 bp extensions homologous to the target sequences (5,7,8,9).
To avoid long residual foreign sequences within modified constructs, FRT or loxP sites flanking selection markers have been used for excision of the positive selection markers with the corresponding recombinases Flp or Cre (6). The persistence of at least one copy of the FRT or loxP sites limits the repeatability of the procedure and may interfere with gene expression in some circumstances. Alternative systems developed for markerless mutagenesis of large DNA sequences in E. coli first insert a cassette consisting of both positive and negative selection markers or bifunctional markers, which allow both positive and negative selection by one gene product. Those cassettes are then replaced with the desired markerless (linear) DNA by a second recombination event and subsequent counterselection (6,10,11,12,13). One problem associated with negative selection markers is that they can be lost unspecifically such as by recombinations between repeat sequences, which are common in both mammalian and viral sequences (data not shown). Methods for markerless replacement without selection markers, such as the use of single oligonucleotides harboring modified sequences (14), the “gene gorging” technique (15), or the “hit-and-fix” protocol (16), lack a powerful procedure to screen for desired recombination events. To raise efficiencies, those methods employ PCR screening of pooled samples (17) or a preselection for recombination events with a marker targeting the E. coli chromosome as a second substrate (18).
The 18-bp recognition site of the homing endonuclease I-SceI was used previously as the negative selection marker with an in vivo cleavage as the counterselection step (10). The technique presented here was refined such that I-SceI is utilized not only for counterselection but also for generation of the substrate for a second Red recombination in E. coli. In this modified strategy, the positive selection markers that were used to introduce the desired target modification in a first step are removed in a second step by the combination of I-SceI cleavage and intramolecular Red recombination utilizing a previously introduced sequence duplication. We show that this modification renders the method versatile and very efficient.Materials and Methods Cells, Viruses, and BAC Clones
Equine herpesvirus type 1 (EHV-1) was propagated on rabbit kidney cells (RK13), maintained in Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Two parental BAC clones were used; both are derived from EHV-1 strain RacL11 in which gene 71 located in the unique-short region was replaced by the mini-F sequence pHA1 (resulting in pL1 1 w) or pHA2 harboring an additional gfp gene within vector sequences (resulting in pL11g; also see Supplementary Figure S1 available online at www.BioTechniques.com) (19,20). Transfection of RK13 cells with pL11w or pL11g DNA isolated from E. coli was used to reconstitute recombinant viruses vL1 1w or vL1 1 g (21).Bacteria and Plasmids
All plasmids were constructed and maintained in E. coli DH10B cells grown at 37°C in Luria Bertani (LB) medium. BAC maintenance and Red recombinations were performed in E. coli strain EL250 [which contains a λ prophage encoding the recombination enzymes Exo, Beta, and Gam (8)] harboring pL11 w or pL11g.
To construct pEPkan-S (see Supplementary Figure S2), the kanamycin resistance gene aphAI derived from plasmid pACYC 177 (New England Biolabs, Ipswich, MA, USA) was amplified by PCR (Supplementary Table S1), adding I-SceI recognition sites and a sequence encoding the FLAG® epitope (Sigma, St. Louis, MO, USA). The PCR product was cut with BamHI and EcoRV and cloned into the BglII and NruI sites of pcDNA3 (Invitrogen, La Jolla, CA, USA).