title: 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.
authors.affiliation: Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, 1801 Fayetteville Street, Durham, NC, 27707, USA, pchatterjee@nccu.edu.
authors.forename: Pradeep K
authors.initials: PK
authors.lastname: Chatterjee
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D017101:Bacteriophage P1 / Q000235:genetics* / Q000378:metabolism; D022202:Chromosomes, Artificial, Bacterial / Q000737:chemistry / Q000235:genetics*; D004251:DNA Transposable Elements; D004742:Enhancer Elements, Genetic; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*; D019895:Transposases / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; C473501:Tn10 transposase; C045073:Cre recombinase; D019426:Integrases; D019895:Transposases