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Generating libraries of iTol2-end insertions at BAC ends using loxP and lox511 Tn10 transposons.

ABSTRACT: Bacterial 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.A 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.The 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.

SUBMITTER: Shakes LA

PROVIDER: S-EPMC3146455 | biostudies-other | 2011 Jul

REPOSITORIES: biostudies-other

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Generating libraries of iTol2-end insertions at BAC ends using loxP and lox511 Tn10 transposons.

Shakes Leighcraft A LA Abe Gembu G Eltayeb Mugtaba A MA Wolf Hope M HM Kawakami Koichi K Chatterjee Pradeep K PK

BMC genomics 20110707

<h4>Background</h4>Bacterial 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 ...[more]

PMID: 21736732

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Project description:BackgroundCitrus species constitute one of the major tree fruit crops of the subtropical regions with great economic importance. However, their peculiar reproductive characteristics, low genetic diversity and the long-term nature of tree breeding mostly impair citrus variety improvement. In woody plants, genomic science holds promise of improvements and in the Citrus genera the development of genomic tools may be crucial for further crop improvements. In this work we report the characterization of three BAC libraries from Clementine (Citrus clementina), one of the most relevant citrus fresh fruit market cultivars, and the analyses of 46.000 BAC end sequences. Clementine is a diploid plant with an estimated haploid genome size of 367 Mb and 2n = 18 chromosomes, which makes feasible the use of genomics tools to boost genetic improvement.ResultsThree genomic BAC libraries of Citrus clementina were constructed through EcoRI, MboI and HindIII digestions and 56,000 clones, representing an estimated genomic coverage of 19.5 haploid genome-equivalents, were picked. BAC end sequencing (BES) of 28,000 clones produced 28.1 Mb of genomic sequence that allowed the identification of the repetitive fraction (12.5% of the genome) and estimation of gene content (31,000 genes) of this species. BES analyses identified 3,800 SSRs and 6,617 putative SNPs. Comparative genomic studies showed that citrus gene homology and microsyntheny with Populus trichocarpa was rather higher than with Arabidopsis thaliana, a species phylogenetically closer to citrus.ConclusionIn this work, we report the characterization of three BAC libraries from C. clementina, and a new set of genomic resources that may be useful for isolation of genes underlying economically important traits, physical mapping and eventually crop improvement in Citrus species. In addition, BAC end sequencing has provided a first insight on the basic structure and organization of the citrus genome and has yielded valuable molecular markers for genetic mapping and cloning of genes of agricultural interest. Paired end sequences also may be very helpful for whole-genome sequencing programs.

Dataset Information

Generating libraries of iTol2-end insertions at BAC ends using loxP and lox511 Tn10 transposons.

Publications

Generating libraries of iTol2-end insertions at BAC ends using loxP and lox511 Tn10 transposons.

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