**1.6 Exogenous DNA cassettes can be introduced precisely at one or both newly created BAC ends**

An important feature of the end-deletion technology is the ease of determining exactly where in the BAC the loxP-transposon had inserted to create the truncation: primers for sequencing the new BAC end have been designed into the loxP and lox511 transposon-ends remaining after the lox-Cre recombinations [see Figure 1 and references (13, 24)], and these have been used to generate ~600 base reads by sequencing the retrofitted BAC DNA directly. An additional feature is the ability to introduce reporter genes and other DNA cassettes precisely at the new end created in the BAC clone. The sequence in front of the loxP or lox511 arrows, as shown in Figures 1 and 4, is retained after the recombination event that creates the deletion [note that orientation of arrow refers to directionality of loxP and lox 511 sequences]. This particular feature has been utilized to place numerous DNA cassettes, such as: i) mammalian cell-selectable antibiotic resistance genes in the BAC (24), ii) a basal promoter-containing EGFP gene in the BAC to serve as an enhancer-trap to functionally localize potential gene-regulatory elements further upstream (14), and iii) introduce iTol2 ends to generate functionalized BAC DNA ready for integration into zebrafish or mouse chromosomes (15).

The ability to truncate either end of a BAC insert progressively while keeping the other end intact is useful to a variety of mapping experiments. Thus genetic markers and polymorphisms have been mapped on a physical map of the chromosome using this technology (13, 16). Long-range gene regulatory elements have also been mapped functionally using the end-deletion approach by either generating transgenic mice with EGFP functionalized BAC DNA (40), or electroporating patient-derived cells in culture (41). The Nkx2-5 gene containing BAC from a mouse library was functionalized with EGFP reporter gene using the phage λ red-recombination system, and then truncated progressively from the far upstream 5' end of the Nkx2-5 gene using a loxP transposon to identify transcriptionenhancing factor binding sites (40). Monitoring mRNA levels of the gene of interest allows circumventing the use of a reporter gene such as EGFP in the BAC or PAC DNA, and a series of upstream deletions in a PAC clone was used to functionally identify potential far-upstream binding sites for transcription factors in the human Hox11 gene (41).

**1.5 Potential Cre-mediated cross-recombination of loxP & lox511 sites does not occur** 

The 34 bp sequence in the mutant lox site, lox511, differs by one nucleotide in the spacer region from that of loxP [see references (24, 32), for actual sequence of lox511]. Varying degrees of promiscuity in recombining different mutant *lox* sites, including the lox511 mutant, with wild type loxP have been reported in previous studies; using both partially purified Cre-extracts *in vitro* (32-36), or Cre over-expressed in cells (36-38). For example, cross recombination between loxP and lox511 has been reported to occur at efficiencies ranging from 5 to 100 % under those experimental conditions that express Cre constitutively (33, 35-38). We have not observed such cross-recombination, and high levels of stringency *in vivo* in recombining identical *lox* sites (21, 24), or *lox* sites with at least identical spacers (39), have been achieved with Cre protein expressed from its native source namely, a phage P1 infection (21, 24, 39). Depending on whether a loxP or a lox511 transposon is used, truncations from the corresponding lox- end can be made readily and exclusively with high stringency [shown schematically in Figure 4]. Thus truncations of genomic DNA from either

**1.6 Exogenous DNA cassettes can be introduced precisely at one or both newly** 

An important feature of the end-deletion technology is the ease of determining exactly where in the BAC the loxP-transposon had inserted to create the truncation: primers for sequencing the new BAC end have been designed into the loxP and lox511 transposon-ends remaining after the lox-Cre recombinations [see Figure 1 and references (13, 24)], and these have been used to generate ~600 base reads by sequencing the retrofitted BAC DNA directly. An additional feature is the ability to introduce reporter genes and other DNA cassettes precisely at the new end created in the BAC clone. The sequence in front of the loxP or lox511 arrows, as shown in Figures 1 and 4, is retained after the recombination event that creates the deletion [note that orientation of arrow refers to directionality of loxP and lox 511 sequences]. This particular feature has been utilized to place numerous DNA cassettes, such as: i) mammalian cell-selectable antibiotic resistance genes in the BAC (24), ii) a basal promoter-containing EGFP gene in the BAC to serve as an enhancer-trap to functionally localize potential gene-regulatory elements further upstream (14), and iii) introduce iTol2 ends to generate functionalized BAC

The ability to truncate either end of a BAC insert progressively while keeping the other end intact is useful to a variety of mapping experiments. Thus genetic markers and polymorphisms have been mapped on a physical map of the chromosome using this technology (13, 16). Long-range gene regulatory elements have also been mapped functionally using the end-deletion approach by either generating transgenic mice with EGFP functionalized BAC DNA (40), or electroporating patient-derived cells in culture (41). The Nkx2-5 gene containing BAC from a mouse library was functionalized with EGFP reporter gene using the phage λ red-recombination system, and then truncated progressively from the far upstream 5' end of the Nkx2-5 gene using a loxP transposon to identify transcriptionenhancing factor binding sites (40). Monitoring mRNA levels of the gene of interest allows circumventing the use of a reporter gene such as EGFP in the BAC or PAC DNA, and a series of upstream deletions in a PAC clone was used to functionally identify potential far-upstream

end are not only efficient, but are highly specific for that end.

DNA ready for integration into zebrafish or mouse chromosomes (15).

binding sites for transcription factors in the human Hox11 gene (41).

**in our method** 

**created BAC ends** 
