Abstract
BackgroundBacterial Artificial Chromosomes (BACs) have been widely used as transgenes in vertebrate model systems such as mice and zebrafish, for a variety of studies. BAC transgenesis has been a powerful tool to study the function of the genome, and gene regulation by distal cis-regulatory elements. Recently, BAC transgenesis in both mice and zebrafish was further facilitated by development of the transposon-mediated method using the Tol2 element. Tol2 ends, in the inverted orientation and flanking a 1 kb spacer DNA (iTol2), were introduced into the BAC DNA within the bacterial host using recombination of homologous sequences. Here we describe experiments designed to determine if a simpler and more flexible system could modify BACs so that they would be suitable for transgenesis into zebrafish or mouse embryos using the Tol2 transposase.ResultsA new technique was developed to introduce recognition sequences for the Tol2 transposase into BACs in E. coli using the Tn10 transposon vector system. We constructed pTnloxP-iTol2kan and pTnlox511-iTol2kan to introduce the loxP or lox511 site and iTol2 cassette, containing the Tol2 cis-sequences in the inverted orientation, into BACs that have loxP and lox511 sites flanking genomic DNA inserts by Tn10-mediated transposition. The procedure enables rapid generation of a large collection of BACs ready for transgenesis with the iTol2 cassette at the new end of a progressively truncated genomic insert via lox-Cre recombination. The iTol2 ends are efficiently recognized by the Tol2 transposase, and the BACs readily integrate into zebrafish chromosomes.ConclusionThe new technology described here can rapidly introduce iTol2 ends at a BAC end of choice, and simultaneously generate a large collection of BACs with progressive deletions of the genomic DNA from that end in a single experiment. This procedure should be applicable to a wider variety of BACs containing lox sites flanking the genomic DNA insert, including those with sequence repeats. The libraries of iTol2 inserted BACs with truncations from an end should facilitate studies on the impact of distal cis-regulatory sequences on gene function, as well as standard BAC transgenesis with precisely trimmed genes in zebrafish or mouse embryos using Tol2 transposition.
Highlights
Bacterial Artificial Chromosomes (BACs) have been widely used as transgenes in vertebrate model systems such as mice and zebrafish, for a variety of studies
In addition to its ease of manipulation, a key advantage of the procedure is the generation of a large collection of BACs with iTol2 introduced at the chosen end of the genomic DNA insert
The results indicate that BACs larger than 100 kb are recognized and processed effectively by the Tol2 transposase, but are integrated efficiently into the zebrafish chromosome when iTol2 is introduced into the BAC DNA ends using a loxP or lox511 Tn10 transposon
Summary
Bacterial Artificial Chromosomes (BACs) have been widely used as transgenes in vertebrate model systems such as mice and zebrafish, for a variety of studies. The Tol transposon system has been used extensively to deliver exogenous DNA into the germline of zebrafish [1,2,3], and more recently for successfully integrating BAC DNA into the germline of both zebrafish and mice [4] In this method the iTol cassette, comprising of the minimal cis-sequences of Tol in an inverted orientation separated by a ~1 kb spacer, was introduced into a BAC. Cre-recombination of the endogenous loxP or lox511 sites, a constituent of all BACs in the public domain, with the ones inserted by these iTol2-Tn10 transposons simultaneously truncates the DNA from the respective end and delivers the iTol cassette. Large numbers of BACs progressively truncated from either end, with iTol placed at the newly created end, can be obtained in a single experiment
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