Directing enhancer-traps and iTol2 end-sequences to deleted BAC ends with loxP- and lox511-Tn10 transposons.
Directing enhancer-traps and iTol2 end-sequences to deleted BAC ends with loxP- and lox511-Tn10 transposons.
复制标题
使用 loxP- 和 lox511-Tn10 转座子将增强子陷阱和 iTol2 末端序列引导至删除的 BAC 末端。
DOI：
10.1007/978-1-4939-1652-8_5
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发表时间：
2015
期刊：
影响因子：
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通讯作者：
Chatterjee,PradeepK
中科院分区：
文献类型：
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作者：
Chatterjee,PradeepK
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-rangecis-acting gene regulatory sequences in these organisms.
