Dataset Information

Characterisation and use of a functional Gadd45g bacterial artificial chromosome.

ABSTRACT: Bacterial artificial chromosomes (BACs) offer a means of manipulating gene expression and tagging gene products in the mammalian genome without the need to alter endogenous gene structure and risk deleterious phenotypic consequences. However, for a BAC clone to be useful for such purposes it must be shown to contain all the regulatory elements required for normal gene expression and allow phenotypic rescue in the absence of an endogenous gene. Here, we report identification of a functional BAC containing Gadd45g, a gene implicated in DNA repair, DNA demethylation and testis determination in mice and exhibiting a broad pattern of embryonic expression. Mouse fetuses lacking the endogenous Gadd45g gene undergo normal testis development in the presence of the Gadd45g BAC transgene. Moreover, a survey of embryonic Gadd45g expression from the BAC reveals that all reported sites of expression are maintained. This functional BAC can now be used for subsequent manipulation of the Gadd45g gene with the confidence that regulatory elements required for embryonic expression, including testis determination, are present. We describe the generation and characterisation of a Gadd45g-mCherry fluorescent reporter exhibiting strong expression in developing gonads and neural tissue, recapitulating endogenous gene expression, as evidence of this.

SUBMITTER: Warr N

PROVIDER: S-EPMC6251886 | biostudies-other | 2018 Nov

REPOSITORIES: biostudies-other

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Characterisation and use of a functional Gadd45g bacterial artificial chromosome.

Warr Nick N May Joel J Teboul Lydia L Suzuki Toru T Asami Maki M Perry Anthony C F ACF Wells Sara S Greenfield Andy A

Scientific reports 20181123 1

Bacterial artificial chromosomes (BACs) offer a means of manipulating gene expression and tagging gene products in the mammalian genome without the need to alter endogenous gene structure and risk deleterious phenotypic consequences. However, for a BAC clone to be useful for such purposes it must be shown to contain all the regulatory elements required for normal gene expression and allow phenotypic rescue in the absence of an endogenous gene. Here, we report identification of a functional BAC c ...[more]

PMID: 30470800

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Project description:The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosis H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosis genome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of approximately 150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovis BCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosis but absent from M. bovis and M. bovis BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosis research projects.

Dataset Information

Characterisation and use of a functional Gadd45g bacterial artificial chromosome.

Publications

Characterisation and use of a functional Gadd45g bacterial artificial chromosome.

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