Isolation and Subcloning of Large Fragments from BACs and PACs

Bacterial artificial chromosome (BAC) vectors and P1-derived artificial chromosome (PAC) vectors are widely used for structural and functional analysis of genomic DNA in vitro and in vivo. The vectors can harbor fragments up to 300 kb in size and show high stability even with alphoid repetitive inserts (7). For structural analysis, they have great advantages because of their stability and low chimerism compared with yeast artificial chromosomes (YACs). For functional analysis in mammalian cells, BACs and PACs often need to be modified for selection and monitoring of the DNA in the cells. There are several refined BAC vectors available for mammalian use (4), but, to use them, the large inserts of BACs and PACs isolated from the major libraries (6) must be recloned into these vectors or new clones must be isolated by screening the new library. BACs can be modified by Cre/loxP recombination (7) or RecET recombination (9), but the most commonly used method is to subclone the insert of a BAC/PAC into a new vector (8). For subcloning, it is necessary to isolate the insert. The isolation of linear BAC fragments is also frequently carried out before introduction into mammalian cells to get stable transformants, as BACs and PACs are circular vectors (2). To isolate and/or separate 100–200 kb linear fragments, the most commonly used protocol is to use pulsed field gel electrophoresis (PFGE) or field inversion gel electrophoresis (FIGE), as for the isolation of YAC fragments (2,5). This method is efficient for isolating pure and intact DNA up to about 2 Mb. However, the method based on PFGE separation is time consuming. Here, we describe an improvement of the method using a mini-gel system with normal electrophoresis that is widely used in laboratories. The modified method is simple, quick, and reliable for isolating large fragments from BACs and PACs. First, we constructed a new BAC vector, named pBeloBAC66D1, which has a mutant loxP (lox66) (1) and neomycin-resistant gene for the purpose of our future experiments (unpublished). The vector has a NotI site for subcloning inserts, which will usually be cut out from available BAC vectors (e.g., pBeloBAC11) (6). All materials were prepared to YAC purification grade as described (5). We used a BAC with the human HPRT gene, bWXD187 (GenBank accession no. AC004383), for vector exchange. To transfer the 155-kb insert of the BAC into the new vector, we digested 2.2 μg BAC DNA with NotI. The digested DNA was applied to a 18 × 6 × 1 mm (length × depth × width) well of a 0.8% low melting point agarose gel (SeaPlaque GTG agarose; BMA, Rockland, ME, USA) and was separated in 1× TAE at 7 V/cm for 45 min without ethidium bromide staining using a Mini-Sub Cell GT (Bio-Rad Laboratories, Hertfordshire, UK). Under these conditions, the large insert fragment (>50 kb) is well separated from the vector fragment (<10 kb), even though linear fragments between 50 and 200 kb are not well separated (Figure 1A). The upper limit to prevent overloading was approximately 2 μg DNA/well (Figure 1B). The DNA fragments were then isolated by agarase treatment essentially as described previously (5) with slight modifications. In brief, a small amount of digested DNA was also loaded to small wells on both sides of the DNA for purification. After electrophoresis, the sides of the gel were cut out and stained with ethidium bromide, and the position of the insert DNA was marked by nicking the agarose. The gels were