Nucleic Acids Research, 2005, Vol. 33, No. 4 e36 doi:10.1093/nar/gni035 Simple and highly efficient BAC recombineering using galK selection Søren Warming, Nina Costantino 1 , Donald L. Court1, Nancy A. Jenkins and Neal G. Copeland* Mouse Cancer Genetics Program and 1Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute, Frederick, MD 21702-1201, USA Received January 11, 2005; Revised and Accepted February 4, 2005 ABSTRACT Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (l) Red-mediated homologous recombination, obviating the need for restriction enzymes and DNA ligases to modify DNA. Here, we describe the construction of three new recombineering strains (SW102, SW105 and SW106) that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection. This two-step selection procedure allows DNA to be modified without introducing an unwanted selectable marker at the modification site. All three strains con- tain an otherwise complete galactose operon, except for a precise deletion of the galK gene, and a defect- ive temperature-sensitive l prophage that makes recombineering possible. SW105 and SW106 cells in addition carry L-arabinose-inducible Cre or Flp genes, respectively. The galK function can be selec- ted both for and against. This feature greatly reduces the background seen in other negative-selection schemes, and galK selection is considerably more efficient than other related selection methods published. We also show how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations, the identification of long-range regu- latory elements and the construction of conditional targeting vectors. INTRODUCTION Bacterial artiﬁcial chromosomes (BACs) have become the DNA of choice for genomic sequencing due to their high stability and large insert size (100–300 kb) (1). BACs are also being used more and more for making transgenic mice, since, in many cases, all of the important regulatory sequences required for normal gene expression can be found on a single BAC (2,3). Many laboratories also use BACs as the starting point for making gene-targeting constructs for manipulating mouse genes using ES cell technology (knock-outs, knock-ins and conditional targeting using Cre/loxP) (4,5). Recombineering (recombination-mediated genetic engi- neering) makes it possible to modify BAC DNA via homo- logous recombination [reviewed in (6,7)]. Recombineering is made possible through the use of three l Red-encoded genes: exo, bet and gam. exo encodes a 50–30 exonuclease that pro- duces 30 overhangs from introduced double-stranded DNA targeting cassettes (dsDNA). bet encodes a pairing protein that binds to the 30 overhangs and mediates its annealing and homologous recombination with complementary DNA present on the BAC. At the same time, gam encodes an inhib- itor of the Escherichia coli RecBCD exonuclease and thereby protects the linear DNA-targeting cassette from degradation by RecBCD. l Red (or the corresponding RecE and RecT genes of the prophage Rac) can be expressed from a multicopy plasmid using an inducible promoter (8,9). Alternatively, these genes can be expressed from a stably integrated defective l prophage, where exo, bet and gam are controlled by the strong phage promoter pL, under stringent control of the temperature-sensitive repressor, cI857 (10,11). In the pro- phage system, exo, bet and gam are not expressed when the bacteria are kept at 32 C. By shifting the bacteria to 42 C for as little as 15 min, the genes are rapidly induced to very high levels and homologous recombination is very efﬁcient. Using recombineering, one can easily subclone a genomic fragment from a BAC by gap repair, either for use as a transgene directly or for subsequent manipulation to make a gene-targeting construct. The introduction of selectable mark- ers into a BAC is also very easy using recombineering. How- ever, a major limitation to the usefulness of BACs is the ease and efﬁciency with which one can make subtle and ‘seamless’ *To whom correspondence should be addressed at Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, West 7th Street at Fort Detrick, Bldg 539, PO Box B, Frederick, MD 21702-1201, USA. Tel: +1 301 846 1260; Fax: +1 301 846 6666; Email: Copeland@ncifcrf.gov ª The Author 2005. 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