Using PAC nested deletions to order contigs and microsatellite markers at the high repetitive sequence containing Npr3 gene locus

Gilmore, Rodney C.; Baker, John; Dempsey, Sean; Marchan, Rosemarie; Corprew, Robert N.L; Byrd, Goldie S.; Maeda, Nobuyo; Smithies, Oliver; Bukoski, Richard D.; Harewood, Ken R.; Chatterjee, Pranam

doi:10.1016/s0378-1119(01)00654-0

Cited by 19 publications

(44 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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]. Such DNA cassettes include sequencing primer binding sites, mammalian cell-selectable antibiotic resistance genes, enhancer-traps and sequences specifically recognized by the vertebrate Tol2 transposase.…”

Section: Strategies For Functionalizing Bacsmentioning

confidence: 99%

“…It is significant that the recombinases used in our approach, Tn10-transposase and Cre protein, respectively, do not act upon repetitive sequences and/or other recombinogenic sites in the genomic DNA insert, therefore minimizing the risk of generating unwanted rearrangement products that are commonly associated with other recombination based methods. Thus BACs spanning loci with highly repetitive DNA content such as those commonly found in the human, and other vertebrate genomes, are also amenable to our methodology (see reference [41] for an example). In contrast to procedures that use sequence homology based recombination to engineer end-deletions, which can only produce one alteration at a time, our approach with loxP or lox511 transposons can generate large collections of end-deleted BACs in a single experiment [41][42][43].…”

Section: Strategies For Functionalizing Bacsmentioning

confidence: 99%

“…Thus BACs spanning loci with highly repetitive DNA content such as those commonly found in the human, and other vertebrate genomes, are also amenable to our methodology (see reference [41] for an example). In contrast to procedures that use sequence homology based recombination to engineer end-deletions, which can only produce one alteration at a time, our approach with loxP or lox511 transposons can generate large collections of end-deleted BACs in a single experiment [41][42][43].…”

Section: Strategies For Functionalizing Bacsmentioning

confidence: 99%

“…Insertions of the Tn10 transposon into BAC DNA from a wide variety of vertebrate genome libraries appear to be random, demonstrating little sequence specificity for transposition [41]. It is unclear whether this lack of sequence specificity arises from the absence of selective pressure evolutionarily for insertions of a prokaryotic transposon into vertebrate DNA, because insertions of Tn10 into prokaryotic DNA have long been known to prefer a somewhat degenerate nevertheless consensus insertion site [44].…”

Section: Strategies For Functionalizing Bacsmentioning

confidence: 99%

“…Note that orientation of arrow refers to directionality of loxP sequence. This feature has been utilized to place numerous DNA cassettes in the BAC, such as: i) mammalian cell-selectable antibiotic resistance genes [41], ii) a basal promoter-containing EGFP gene to serve as an enhancer-trap to functionally locate potential generegulatory elements further upstream [40], and iii) introduce end sequences of the vertebrate transposn iTol2 to generate integration-ready GFP-functionalized BAC DNA for zebrafish or mouse transgenesis studies [43,62].…”

Section: Introducing Exogenous Dna Cassettes Precisely At One or Bothmentioning

confidence: 99%

See 4 more Smart Citations

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

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Strategies For Functionalizing Bacsmentioning

confidence: 99%

Section: Strategies For Functionalizing Bacsmentioning

confidence: 99%

Section: Strategies For Functionalizing Bacsmentioning

confidence: 99%

Section: Strategies For Functionalizing Bacsmentioning

confidence: 99%

Section: Introducing Exogenous Dna Cassettes Precisely At One or Bothmentioning

confidence: 99%

See 3 more Smart Citations

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

Self Cite

View full text Add to dashboard Cite

show abstract

Directing Enhancer-Traps and iTol2 End-Sequences to Deleted BAC Ends with loxP- and lox511-Tn10 Transposons

Chatterjee

2014

Methods in Molecular Biology

View full text Add to dashboard Cite

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-range cis-acting gene regulatory sequences in these organisms.

show abstract

Selecting transpositions using phage P1 headful packaging: new markerless transposons for functionally mapping long-range regulatory sequences in bacterial artificial chromosomes and P1-derived artificial chromosomes

Chatterjee¹,

Mukherjee²,

Shakes³

et al. 2004

Analytical Biochemistry

View full text Add to dashboard Cite

Using PAC nested deletions to order contigs and microsatellite markers at the high repetitive sequence containing Npr3 gene locus

Cited by 19 publications

References 20 publications

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

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

Directing Enhancer-Traps and iTol2 End-Sequences to Deleted BAC Ends with loxP- and lox511-Tn10 Transposons

Selecting transpositions using phage P1 headful packaging: new markerless transposons for functionally mapping long-range regulatory sequences in bacterial artificial chromosomes and P1-derived artificial chromosomes

Contact Info

Product

Resources

About