**1. Introduction**

44 Bacterial Artificial Chromosomes

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F., Tassabehji, M., & Shaffer, L.G. (1998) Delineation of the common critical region in Williams syndrome and clinical correlation of growth, heart defects, ethnicity, Strategies for altering sequences in large DNA inserts in BACs are fundamentally different from those traditionally used for small plasmids. Two factors are primarily responsible for this: the existence of a high multiplicity of sites in BACs recognized by DNA modifying enzymes, such as restriction endo-nucleases, as well as the brittle nature of large duplex DNA that is not packaged with DNA-binding proteins in the test-tube. The large number of DNA fragments generated by the common restriction enzymes, and the unavailability of robust separation techniques to isolate and keep track of the relative order of the pieces; precludes using the "cut-and-paste" mechanism to alter DNA sequences in BACs. Instead DNA recombination *in vivo* has become the method of choice for modifying BACs. Although the net result again is cutting and re-joining of DNA, the entire process is concerted in the bacterial host with no free ends of DNA to go astray; and occurs primarily in a nucleoprotein complex that protects it from shear forces which otherwise would break the large DNA during manipulations *in vitro.*

Two separate approaches based upon using sequence homology for recombining DNA were developed independently to alter sequences in BACs. The first method introduces the major recombination function of *E.coli*, RecA, back into the severely recombination deficient host DH10B, originally engineered to propagate vertebrate DNA in BACs (1). The second approach introduces recombination functions of phage λ, namely red α, red β and red γ into the DH10B host, and utilizes recombination of homologous sequences of shorter length to insert exogenous DNA cassettes into BACs (2). Both methods have been widely used to engineer a variety of alterations in BAC DNA, such as introducing reporter gene cassettes into the genomic insert, mutating sequences at a target site, and introducing loxP sites (1-11).

A different strategy for modifying BAC DNA also uses recombination, but does not require targeting vectors to carry sequences homologous to those in the genomic insert to introduce exogenous DNA cassettes. Insertions of the bacterial Tn10 transposon can introduce exogenous DNA, including lox sites, at random locations in the BAC (12, 13). Site-specific recombination using the Cre-lox system on the other hand, can deliver reporter genes and other exogenous DNA cassettes such as sequencing primer sites, mammalian cell-selectable antibiotic resistance genes, enhancer-traps and sequences specific to the vertebrate transposon Tol2 precisely at the ends of the genomic DNA insert in BACs (14-16). It is significant that the recombinases involved in either of these approaches, Tn10-transposase and Cre protein respectively, do not act upon sequence repeats and/or other recombinogenic sites in the genomic DNA insert to rearrange it. This particular characteristic should make the approach applicable to a wider variety of BACs in the public domain, including those containing repetitive sequences [see reference (16) for an example].

Insertions of the Tn10 transposon into DNA of BACs from a wide variety of vertebrate genome libraries appear to be random, demonstrating little sequence specificity for transposition (16). It is unclear whether this lack of sequence specificity arises from the absence of selective pressure evolutionarily for insertions of a prokaryotic transposon into vertebrate DNA, because insertions into prokaryotic DNA have long been known to prefer a somewhat degenerate nevertheless consensus insertion site (17). The minor sequence preferences for insertions of Tn10 observed in BACs probably have more to do with availability of sites for Tn10 insertions in HU protein-packaged vertebrate DNA in the bacterium than specificity for sequences. On occasion, incorporation of Tn10 into insert DNA of a rare BAC clone displays apparent sequence selectivity (18). However, this was clearly shown to be due to a clonal selection process that arose from picking single colonies of transposon plasmid transformed BAC clones which had induced the transposase gene prior to actual induction with Isopropyl β-D-1-thiogalactopyranoside (IPTG). Slight modification of the procedure, that has since recommended inducing a large pool of transposon plasmid transformed BAC colonies instead of a single one, has rectified this potential problem completely (18). A detailed description of the transposon retrofitting approach for BACs follows.
