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The generation of transgenic animals using a promoter to direct the expression of a gene of interest is a continuously developing technique. Traditionally, a short fragment from the promoter of a gene with the desired expression profile (plasmid-based transgene) has been used as a vector to express a gene of interest. However, more recently, large pieces of DNA such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and P1-based artificial chromosomes (PACs) are replacing plasmid-based transgenes, which is due mainly to three reasons: (i) large pieces of DNA are more likely to contain all of the promoter elements needed for faithful gene expression; (ii) expression of the gene of interest is more likely to be copy number dependent; and (iii) there is less variability in the pattern of expression across transgenic lines (2,3). Recently, the modification of BACs and PACs has become a routine technique (5,6). Usually, the gene of interest is placed downstream of the ATG of a chosen gene in the BAC or PAC. The rationale for using BACs and PACs is that these large fragments of DNA are likely to contain all or most of the cis elements that regulate the expression of the gene chosen to drive the expression of the transgene. Therefore, it is of obvious importance to know the localization of the gene chosen for expression within the BAC or PAC, as a localization too proximal to one of the ends can result in a loss of some regulatory elements upstream or downstream of the ATG needed to drive faithful gene expression. This orientation can be achieved by restriction analysis, which is usually difficult and cumbersome. Here, we present a straightforward method that allows simultaneous modification of a BAC or PAC and the localization of the gene within the BAC or PAC in a single step. For this purpose, we generated an expression cassette containing the following features: the gene to be expressed (here the iCre recombinase), a codonimproved Cre recombinase (4) with a polyadenylation signal from bovine growth hormone (BD Biosciences Clontech, Palo Alto, CA, USA) and an ampicillin resistance cassette flanked by two Flp recombinase target (FRT) sites and containing an internal NotI restriction site to allow the localization of the gene within the PAC or BAC (Figure 1). A 120-kb PAC containing the Kainic receptor 1 gene (KA1) was isolated for use as a vector to drive the expression of the iCre recombinase. The K A1 PAC was modified by ET-recombination (homologous recombination in E. coli) as previously described (6). Briefly, a 150-bp homology arm to the K A1 gene was amplified by PCR and cloned in front of the iCre recombinase, replacing the internal ATG of the KA1 receptor gene with the iCre ATG. A 3′ homology region to the KA1 gene of 150 bp was amplified by PCR and cloned at the end of the construct (Figure 1). DH10B bacteria containing the K A1 PAC were transformed with a tetracycline-resistant version of the plasmid pBAD ETγ [plasmid that contains the necessary elements to carry out homologous recombination and modification of the PAC (6)]. The cells were induced with arabinose (0.2%), and competent cells were generated. The digested targeting vector was electroporated into competent cells previously induced with arabinose and containing the KA1 PAC. Putative positive colonies believed to have undergone homologous recombination were selected in the presence of kanamycin (PAC vector resistance) and ampicillin. Homologous recombination of the targeting construct with the appropriate sequences in the PAC was confirmed by restriction analyses, Southern analyses, and PAC sequencing. The frequency of correct targeted PACs was 8 of 10 (i.e., 8 of 10 colonies that had undergone recombination had the iCre cassette inserted in the right place). The localization of the KA1 gene within the PAC was performed as follows. The modified PAC KA1 was subjected to NotI digestion and analyzed by pulsed-field gel electrophoresis (Figures 1 and 2). The size-separated DNA was transferred to nylon membrane and probed with a radiolabeled oligonucleotide immediately upstream Benchmarks

Rapid Localization of a Gene within BACs and PACs