Isolating large nested deletions in bacterial and P1 artificial chromosomes by in vivo P1 packaging of products of Cre-catalysed recombination between the endogenous and a transposed loxP site

Chatterjee, Pranam; Coren, Jonathon S.

doi:10.1093/nar/25.11.2205

Cited by 32 publications

(35 citation statements)

References 24 publications

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“…The virulent form of P1 phage (P1 vir), the bacterial Cre Ϫ host used to transfer PAC͞BAC-nested deletions (Escherichia coli strain NS3516), and procedures to grow and titer P1 phage have been described (9)(10)(11). Super Broth (Advanced Biotechnologies, Columbia, MD) was used to grow BAC͞PAC deletion clones to high cell densities for plasmid DNA isolation.…”

Section: Methodsmentioning

confidence: 99%

“…Transposon plasmids pTnBAC͞loxP or pTnPGKpuro͞loxP described earlier were introduced into a BAC or PAC clone, respectively, by calcium chloride transformation (9,12). Procedures for inducing transposon insertions, transduction of retrofitted BAC or PAC plasmid with P1 phage, and isolating DNA carrying nested deletions have been described (9,10) with the exception that 2 mM IPTG was used to induce transposition in our current study.…”

Section: Methodsmentioning

confidence: 99%

“…We have used a loxP sequence containing transposon 10 (Tn10) minitransposon insertion strategy (retrofitting) to generate nested deletions from one end of the genomic insert in BACs and PACs (9). A small, transposon-containing plasmid is introduced into the clone of interest, followed by a single-step and single-tube process for generating deletions in vivo.…”

mentioning

confidence: 99%

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Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms

Chatterjee

Yarnall²,

Haneline³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205-2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable. I dentifying polymorphic sites in the genome is a basic aspect of molecular genetics and genomics. The process is needed for a variety of purposes, ranging from the development of polymorphic marker sets useful as a tool for genetic analysis of a chromosomal region or full genome scan, to the initial identification of variants or mutations in a newly discovered gene (1, 2). In most cases, the identity of base differences and their location relative to a gene or other polymorphic sites is either useful or required. Recent estimates of the number of singlenucleotide polymorphisms (SNPs) needed for whole genome association studies in humans vary from several thousand to several hundred thousand (1, 3); thus, efficient and cost-effective methods for identifying a large number of SNPs with the required characteristics of dense yet even spacing, and of known order over large uncharacterized regions of the genome, is of interest. A comparison of two methods to develop a densely ordered map of SNPs covering a 4-Mb region of the human genome was recently reported (4). In one approach, large-insert bacterial clones, bacterial artificial chromosomes (BACs) (5) and P1 artificial chromosomes (PACs) (6), spanning this region were fragmented and reconstructed in 2-kb plasmid libraries, which were then sequenced. This shotgun procedure is efficient in identifying SNPs; however, to approach a map of 30-kb average SNP spacing, bidirectional sequencing of approximately 500 randomly chosen subclones per 100 kb of genomic sequence was required. Multiple BAC and PAC clones mapping to the region...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms

Chatterjee

Yarnall²,

Haneline³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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“…Germ-line expression of the modified BAC in zebrafish or mice has been achieved with yet a third recombination system, namely the vertebrate Tol2 system, described later in the article. Insertions of the bacterial transposon Tn10 can introduce exogenous DNA, including lox sites, at random locations in BACs [39,40]. Site-specific recombination by the Cre-lox system on the other hand, can deliver these reporter genes and other exogenous DNA precisely at the ends of genomic DNA inserts in BACs [41][42][43].…”

Section: Strategies For Functionalizing Bacsmentioning

confidence: 99%

Long range gene-regulatory sequences identified by transgenic expression of bacterially-engineered enhancer-trap BACs in zebrafish

Wolf¹,

Nyabera²,

Torre³

et al. 2014

Mol Biol Genet Eng

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Large pieces of DNA from the chromosomes of numerous organisms, including the human, are faithfully propagated in bacteria as large extra-chromosomal plasmids known as Bacterial Artificial Chromosomes (BACs). Because they represent tiny contiguous pieces of the chromosome, BACs are ideally suited for expression of genes in their chromosomal contexts. Genes in BACs need to be functionalized with reporter genes and other sequences that allow easy monitoring, stable maintenance and propagation of the DNA in the new host organism. BAC DNA can be altered within its bacterial host in several ways. One approach uses Tn10 mini-transposons to introduce exogenous DNA into BACs for a variety of purposes. The random insertions of Tn10 transposons carrying lox sites have helped position mammalian cellselectable antibiotic resistance genes, enhancer-traps and inverted repeat end-sequences of the vertebrate transposon Tol2 precisely at the ends of genomic DNA inserts in BACs. Functional identification of generegulatory elements through reporter gene expression and BAC DNA integration into zebrafish or mouse chromosomes have been facilitated with such retrofitting. The methodology has been used extensively to dissect the regulation of the Amyloid Precursor Protein (appb) gene in zebrafish. Functional identification of long-range regulatory sequences of appb has provided important clues for regulation of the APP gene in humans.

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“…It does not require sequence homology between vector and genomic inserts of BACs to introduce exogenous DNA cassettes. Insertions of the bacterial transposon Tn10 can introduce exogenous DNA, including lox sites, at random locations in BACs [56,57]. Furthermore, site-specific recombination by the Cre-lox system can deliver these reporter genes and other exogenous DNA precisely to the ends of genomic DNA inserts in BACs [58][59][60].…”

Section: B) Transposition Followed By Progressive Deletions From Bacendsmentioning

confidence: 99%

Trapping Enhancers by Transgenic Expression of BACs Engineered in Bacteria with loxP Transposons

Nyabera¹

2014

Int J Genomic Med

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Bacterial Artificial Chromosomes (BACs) are large extra-chromosomal plasmids in bacteria that faithfully propagate large pieces of DNA from the chromosomes of organisms. Because they represent tiny contiguous pieces of the chromosome, BACs are ideally suited for expression of genes in their chromosomal contexts. Genes in BACs can be monitored for expression after the DNA is modified with reporter genes and other sequences that allow it to be stably propagated in the new host. Several methods have been developed to alter BAC DNA within its bacterial host. One approach uses Tn10 mini-transposons to introduce exogenous DNA into BACs. The random insertions of Tn10 carrying lox sites have directed mammalian cell-selectable antibiotic resistance genes, enhancer-traps and inverted repeat ends of the vertebrate transposon Tol2 precisely to the ends of genomic DNA inserts in BACs. Reporter gene expression from BAC DNA integrated into zebrafish or mouse chromosomes have resulted from such retrofitting. The methodology has been used extensively to analyze regulation of the Amyloid Precursor Protein (appb) gene in zebrafish. Functional identification of long-range regulatory sequences of appb has provided important clues for regulation of the APP gene in humans.

show abstract

Isolating large nested deletions in bacterial and P1 artificial chromosomes by in vivo P1 packaging of products of Cre-catalysed recombination between the endogenous and a transposed loxP site

Cited by 32 publications

References 24 publications

Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms

Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms

Long range gene-regulatory sequences identified by transgenic expression of bacterially-engineered enhancer-trap BACs in zebrafish

Trapping Enhancers by Transgenic Expression of BACs Engineered in Bacteria with loxP Transposons

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