Directing Enhancer-Traps and iTol2 End-Sequences to Deleted BAC Ends with loxP- and lox511-Tn10 Transposons

Abstract

A step-by-step detailed procedure is presented to progressively truncate genomic DNA inserts from either end in BACs. The bacterial transposon Tn10 carrying a loxP or a lox511 site is inserted at random into BAC DNA inside the bacterial cell. The cells are then infected with bacteriophage P1. The Cre protein expressed by phage P1 generates end-deletions by specifically recombining the inserted loxP (or lox511) with the loxP (or lox511) endogenous to and flanking insert DNA in BACs from the respective end. The Cre protein also helps phage P1 transduce the BAC DNA by packaging it in P1 heads. This packaging by P1 not only recovers the rare BAC clones containing Tn10 insertions efficiently but also selects end-truncated BACs from those containing inversions of portions of their DNA caused by transposition of Tn10 in the opposite orientation. The libraries of end-deleted BACs generated by this procedure are suitable for numerous mapping studies. Because DNA in front of the loxP (or lox511) arrowheads in the Tn10 transposon is retained at the newly created BAC end, exogenous DNA cassettes such as enhancer-traps and iTol2 ends can be efficiently introduced into BAC ends for germline expression in zebrafish or mice. The methodology should facilitate functional mapping studies of long-range cis-acting gene regulatory sequences in these organisms.

Trapping Enhancers by Transgenic Expression of BACs Engineered in Bacteria with loxP Transposons

“…Cre protein acts in trans to also recombine the loxP site transposed into the BAC DNA insert with the loxP endogenous to BAC vector and located at one end of insert DNA (Figure 2, Panel D). Thus the transposed loxP of one orientation produces a deletion from one end of genomic insert, while the loxP inserted in the opposite orientation simply inverts the DNA between it and the one endogenous to the BAC [63].…”

“…The DNA in BAC clones is isolated by simple mini-prep procedures, digested with Not I enzyme and analyzed by FIGE [63,64]. High throughput formats for preparing DNA from BAC deletions in 96-well plates, suitable for subsequent FIGE analyses and end sequencing, have also been described [57,64].…”

“…While generating mutations in the small Tnplasmid is rapid, creating and characterizing an enhancer-trap BAC deletion library with the mutant transposon is not. It takes about a month by an individual to get a set of four BAC libraries done [63]. Expression analyses of selected clones from these 18 libraries indicated binding sites of at least two known transcription factors are important for function.…”