**2.4 Pros and cons of the transposon retrofitting approach**

The insertions of exogenous DNA cassettes into BAC DNA are performed using Tn10 transposons carrying loxP or lox511 sites, and do not rely upon sequences existing in the genomic inserts of BACs. Consequently, the Tn10 transposons developed for a particular objective are applicable to all BACs in the public domain. This feature is unlike the targeting vectors used in homologous recombination dependent strategies, where the targeting vector plasmids need to be constructed anew for each BAC.

Because the insertions of these Tn10 transposons into genomic DNA in BACs appear to be largely random [see references (16) for discussion], large collections of BACs with ends

[reference (14), and Shakes, Du, Sen, Abe, Kawakami, Wolf, Hatcher, Norford and

The BAC enhancer-trap technology described above has three additional features that should prove beneficial: i) allows sampling much larger sequences of DNA, and consequently multiple discontinuous regulatory domains simultaneously compared to small plasmids, ii) the context of regulatory DNA with respect to the gene and chromosome is preserved and, iii) can be used with BACs in established libraries from a wide variety of organisms, and tested in several species. Although the methodology does not allow generation of internal deletions, truncations from the end opposite to the enhancer-trap can be made with a lox511 transposon [as seen when iTol2-end DNA cassettes were introduced at the other end of BAC DNA, (Figures 5 and 6)] to explore functions of candidate regulatory regions in a limited way. Sequences bending DNA (45-47), or phasing nucleosomes and other transcription factors (48-50) are left unaltered using BACs compared to characteristics of the gene region found endogenously. Bringing exogenous pieces of DNA together to create artificial junctions in small plasmids to trans-activate reporter genes does not adequately address the endogenous role of the regulatory sequence; and this is avoided using BACs. It is probably worth noting that DNA structure surrounding regulatory factor binding sites have evolved over long periods, and these are also left unaltered here. We noted earlier that there were 52 sites with six or more consecutive Aresidues, known to cause bends in unpackaged DNA (46), in the ~28 kb upstream

In a different application Tn10 transposons carrying 'arm' mutants of loxP, such as lox66, have been used to successfully substitute the loxP site at one end of the genomic insert DNA in a BAC (39). It should be possible also to substitute the lox511 site at the other end of insert DNA in a similar manner using a lox site with an identical 'spacer' as lox511 but carrying a different 'arm' sequence. The usefulness of such substitutions of loxP or lox511 sites in BACs with lox66 is realized from the fact that the integration of loxP plasmids to a loxP site on the chromosome is reversible, with the equilibrium favored for excision. Targeting lox66 substituted BACs specifically to pre-positioned lox71 sites in chromosomes via lox-Cre recombination should be irreversible. Such strategies are likely to be particularly useful in systems such as the zebrafish, where classical "knock-in" technology cannot be used because of genome duplication in an ancestral teleost [see reference (39) for discussion].

The insertions of exogenous DNA cassettes into BAC DNA are performed using Tn10 transposons carrying loxP or lox511 sites, and do not rely upon sequences existing in the genomic inserts of BACs. Consequently, the Tn10 transposons developed for a particular objective are applicable to all BACs in the public domain. This feature is unlike the targeting vectors used in homologous recombination dependent strategies, where the targeting vector

Because the insertions of these Tn10 transposons into genomic DNA in BACs appear to be largely random [see references (16) for discussion], large collections of BACs with ends

regulatory sequence identified as important in regulating APPb (14).

**2.3 Substitution of Lox sites flanking BAC inserts with Lox66** 

**2.4 Pros and cons of the transposon retrofitting approach** 

plasmids need to be constructed anew for each BAC.

Chatterjee manuscript in preparation].

progressively trimmed are generated in a single experiment. Such libraries, when made with Tn10 transposons carrying enhancer-traps or iTol2 cassettes, are uniquely suited for functionally mapping long-range gene regulatory sequences using transgenic animals. Thus multiple BACs from a contig spanning a genetic locus should enable such functional analyses to be extended over large sections of the genome. The approach does not require selecting sequences for mutational analysis to test their gene regulatory potential, thus it is an unbiased approach. It enables enhancer-trap containing BACs, or BACs retrofitted with EGFP cassettes by sequence homology based recombination, to be converted into deletion libraries with integrated iTol2 ready for chromosome integration. Thus compared to traditional approaches for enhancer-trapping used with whole genomes in animals (51-59), our approach using individual BACs, has the potential to allow a more uniform coverage of the genome because the baseline efficiency of trap insertion is reset for individual BACs in the bacterial host. Although there appear to be vast regions of the genome refractory to enhancer-trapping by traditional means, to date we have not encountered BACs refractory to Tn10 transposon insertions.

The absence of undesirable rearrangement of insert DNA due to the high content of sequence repeats in certain BACs is an additional advantage with our approach. We note that vertebrate DNA in general and mammalian DNA in particular, has large numbers of DNA sequences that are repeated (16). Use of radioactive isotopes is also avoided because no Southern blotting is required in retrofitting BACs with transposons.

A notable drawback of the transposon based approach appears to stem from the very feature that makes it readily doable: the P1 headful packaging strategy used to isolate the functionalized BAC so easily also limits the size of BAC DNA that can be analyzed to ~ 110 kb. Although the genomic DNA insert is truncated in the process, the resulting 103 kb insert DNA size, the remainder being modified BAC vector, is unlikely to be a disadvantage in most applications; because a majority of vertebrate genes can be housed in their entirety within this size limit. Almost half of evolutionarily conserved non-coding gene-regulatory sequences in vertebrate genomes (60), and probably a similar fraction of those that are conserved in function and shape but not in sequence (43, 44), are located within this span of DNA adjoining start sites of transcription of genes.

Lastly, both the lox sites flanking insert DNA in BACs can be readily substituted with lox sites with identical spacer but different arms using Tn10 transposons (39). Such replacements would make the ~110 kb retrofitted BACs amenable to "knock-in" strategies of a slightly different type: BACs with lox66 substituted for loxP should be targetable to lox71 sites in chromosomes of organisms such as zebrafish, a system where conventional homologous recombination-mediated "knock-in" technology is unavailable due to genome duplication in an ancestral teleost during evolution (61).

Clearly the transposon retrofitting strategy and those based on homologous recombination have strengths that appear somewhat complimentary in nature. Thus a judicious approach might be to use a combination of the two methodologies, as demonstrated in earlier studies (40, 15). Introducing the Tol2 inverted repeat ends to the ends of genomic DNA insert in a BAC that was previously functionalized with a reporter gene appears straightforward and easier to do with the Tn10 transposon approach. The libraries of iTol2 inserted BACs generated should also facilitate integration of trimmed single genes into the germline and help to functionally map *cis*-acting gene regulatory sequences in animals. The approach should be applicable to a wider variety of BACs, including those with sequence repeats.
