**1.1 Methodology for Tn10 transposon retrofitting of BACs**

The Tn10 transposon modification procedure for BACs is conceptually simple, as illustrated in Figure 1. A loxP sequence is placed within the 70 base inverted repeat ends of the bacterial Tn10 mini-transposon (shown in the lower panel of Figure 1).

The plasmid DNA carrying the transposon is introduced into the same cell that houses the BAC plasmid and the transposase gene, located outside the inverted repeat ends in the transposon plasmid, is induced. Upon induction the transposase protein excises the DNA cassette flanked by the inverted repeats (shown as green and pink boxes L & R in the transposon plasmid) and inserts it into other nearby DNA in the bacterial host, including the bacterial genome and the BAC DNA. Insertions of the Tn10 into BAC DNA occur in either orientation, and are irreversible because the transposase gene is left behind in the excised transposon plasmid and destroyed. Important considerations that dictate subsequent steps in the procedure include 1) damage to the host genome from Tn10 insertions, and 2) the fraction of BACs actually modified is relatively low ~ 1 in 10,000. Therefore efficient steps are required to recover the low percentage of BACs containing insertions and subsequently transfer these into a new host. Both these challenges are met by packaging the Tn10 modified BAC DNA in phage P1 heads. Therefore the cells containing the BAC DNA with

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.

**1.1 Methodology for Tn10 transposon retrofitting of BACs** 

bacterial Tn10 mini-transposon (shown in the lower panel of Figure 1).

The Tn10 transposon modification procedure for BACs is conceptually simple, as illustrated in Figure 1. A loxP sequence is placed within the 70 base inverted repeat ends of the

The plasmid DNA carrying the transposon is introduced into the same cell that houses the BAC plasmid and the transposase gene, located outside the inverted repeat ends in the transposon plasmid, is induced. Upon induction the transposase protein excises the DNA cassette flanked by the inverted repeats (shown as green and pink boxes L & R in the transposon plasmid) and inserts it into other nearby DNA in the bacterial host, including the bacterial genome and the BAC DNA. Insertions of the Tn10 into BAC DNA occur in either orientation, and are irreversible because the transposase gene is left behind in the excised transposon plasmid and destroyed. Important considerations that dictate subsequent steps in the procedure include 1) damage to the host genome from Tn10 insertions, and 2) the fraction of BACs actually modified is relatively low ~ 1 in 10,000. Therefore efficient steps are required to recover the low percentage of BACs containing insertions and subsequently transfer these into a new host. Both these challenges are met by packaging the Tn10 modified BAC DNA in phage P1 heads. Therefore the cells containing the BAC DNA with transposon insertions are infected with P1 phage after induction of the transposase gene with IPTG [see reference (19) for a detailed description of the procedure].

Fig. 1. Legend: Schematic representation of the BAC end-deletion technology using a loxP transposon. The inverted triangle represents the loxP transposon, which is shown in greater detail in the lower panel. The 70 bp inverted repeat ends of the transposon are indicated by the green and pink boxes marked L and R respectively. The thick black arrow represents the loxP site in both the transposon and BAC vector DNA. The broken arrow represents the lox511 site in BAC vector. Because transposon insertions are rare, single random transpositions into the BAC DNA occur when the transposase gene is induced. Upon Cre recombination, the transposon-inserted loxP of identical orientation to the loxP endogenous to the BAC generates a deletion of the DNA between them. Inversions are not shown. The pool of BAC DNA molecules therefore generates a library of end-deleted BACs from the random insertions of a loxP transposon into BAC DNA molecules.

Infection with P1 phage serves an additional purpose. The phage expresses Cre protein early in its life cycle to circularize the otherwise linear DNA within the phage head. Newly synthesized Cre protein acts *in trans* to also recombine the loxP site transposed into the BAC genomic insert with the loxP endogenous to the BAC and located at one end of the insert DNA [see step 1, Figure 2]. Thus the transposed loxP site of one orientation produces a deletion from one end of the genomic insert, while the loxP inserted in the opposite orientation simply inverts the DNA between it and the one endogenous to the BAC. Because BACs in all modern libraries carry insert DNA of average size ~ 160 kb, and because the amount of DNA that can be packaged in a P1 head is ~110 kb (20), the P1 headful packaging step can serve as a selection strategy to isolate transpositions of loxP in the orientation identical to the endogenous one (12), as only that allows reducing the BAC DNA length to less than ~110 kb. Only those end-deletions that reduce the length of BAC DNA to less than ~110 kb are rescued, as shown in Figure 2.

Fig. 2. Legend: Schematic representation of the DNA recombinations occurring in the transposon retrofitting of BACs. Step 1 shows creation of a deletion in the BAC DNA by Cre recombination of the transposed and endogenous loxP sites. Step 2 shows the Cre recombination of circularized phage P1 DNA (P, G, E) with the end-deleted BAC DNA (B, A, C) to generate the co-integrate. The phage packaging site "pac site" and the Cre gene are indicated in the phage DNA by the thick solid bar and the X, respectively. Step 3 shows the BAC DNA packaged in the phage P1 head, its recombination after entry into cells by newly synthesized Cre protein, and circularization into a BAC plasmid. If the length of DNA (B, A, C) exceeds the headful packaging capacity of ~110 kb, then the second loxP site (indicated by the thick arrows) would not fit in the phage head and the DNA cannot circularize by loxP-Cre recombination after entry into cells. This leads to the BAC DNA not being rescued. Note that the Cre gene is also lost upon circularization of the truncated BAC DNA.

orientation simply inverts the DNA between it and the one endogenous to the BAC. Because BACs in all modern libraries carry insert DNA of average size ~ 160 kb, and because the amount of DNA that can be packaged in a P1 head is ~110 kb (20), the P1 headful packaging step can serve as a selection strategy to isolate transpositions of loxP in the orientation identical to the endogenous one (12), as only that allows reducing the BAC DNA length to less than ~110 kb. Only those end-deletions that reduce the length of BAC DNA to less than

Fig. 2. Legend: Schematic representation of the DNA recombinations occurring in the transposon retrofitting of BACs. Step 1 shows creation of a deletion in the BAC DNA by Cre

recombination of circularized phage P1 DNA (P, G, E) with the end-deleted BAC DNA (B, A, C) to generate the co-integrate. The phage packaging site "pac site" and the Cre gene are indicated in the phage DNA by the thick solid bar and the X, respectively. Step 3 shows the BAC DNA packaged in the phage P1 head, its recombination after entry into cells by newly synthesized Cre protein, and circularization into a BAC plasmid. If the length of DNA (B, A, C) exceeds the headful packaging capacity of ~110 kb, then the second loxP site (indicated by the thick arrows) would not fit in the phage head and the DNA cannot circularize by loxP-Cre recombination after entry into cells. This leads to the BAC DNA not being rescued.

recombination of the transposed and endogenous loxP sites. Step 2 shows the Cre

Note that the Cre gene is also lost upon circularization of the truncated BAC DNA.

~110 kb are rescued, as shown in Figure 2.

Packaging of the end-deleted BAC DNA occurs from a large co-integrate plasmid in the cell which is described in detail elsewhere (21, 22). In co-integrate formation, the P1 phage DNA effectively contributes a packaging location named the "pac site" which is recognized by the phage packaging proteins [shown in step 2, Figure 2]. The co-integrate DNA is thought to replicate by a "rolling circle" mechanism. A cut is made in the newly synthesized co-integrate DNA at the "pac site", and the DNA end corresponding to piece loxP-B, A, C is stuffed into newly assembled empty heads of phage P1. Packaging of DNA from co-integrate is directional and the second cleavage is made after the P1 head is full regardless of sequence using a "headful-cleavage" mechanism (21, 22). Note that if Cre recombination of the transposed loxP with the loxP endogenous to the BAC did not reduce the length of BAC DNA segment B, A, C, to less than ~110 kb, this piece of BAC DNA inside the phage head would not be flanked by loxP on both ends [see step 3 of Figure 2]. In the absence of loxP sites flanking BAC DNA on both ends, the DNA would be unable to circularize by Cre recombination and would be destroyed when introduced into cells. Insertions of loxP in the other orientation, that cause inversion of the DNA, do not reduce the size of segment B, A, C to less than 110 kb. Consequently, the BAC DNA in P1 heads from inversions is destroyed upon entry into cells.

The bacterial lysate containing P1 heads packaged with end-deleted BAC DNA is treated with chloroform. This treatment not only facilitates lysis of P1 infected recombination deficient DH10B but also kills all cells harboring intact transposon and BAC plasmids. This killing is important for the selection of transposon inserted BACs in the next stage when antibiotic selection is employed, as otherwise cells without transposon insertions into BACs but merely carrying the two intact plasmids would get selected. The lysate after chloroform treatment is used to infect fresh bacteria. Upon entry of the phage packaged BAC DNA into cells; Cre protein is expressed from the DNA end adjacent to the second loxP site [indicated as X in phage head and co-integrate], but only transiently, because the Cre gene is lost after Cre-loxP recombination to circularize the linear BAC DNA [Step 3, Figure 2]. Cells are plated on LB plates containing chloramphenicol to select for clones of end-deleted BAC. Note that the phage DNA segment [P, G, E] from the co-integrate is incompletely packaged, if at all, and is destroyed after the loxP-Cre circularization in the new host. Even the rare phage particle containing a complete phage genome is unlikely to replicate or survive because of the chloramphenicol selection on the plates.

Selecting with a single antibiotic for the BAC DNA is sufficient for the first round of enddeletions, using either a loxP or a lox511 transposon. Additional selection for transposition of lox sites is not necessary in this first round because the P1 headful packaging itself serves as the selection for the low frequency of lox-site transpositions in the previous step [see references (23, 24) for detailed discussions].
