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

Chatterjee, Pranam; Mukherjee, Sushmita; Shakes, Leighcraft A.; Wilson, Willie; Coren, Jonathon S.; Harewood, Ken R.; Byrd, Goldie S.

doi:10.1016/j.ab.2004.09.016

Cited by 11 publications

(47 citation statements)

References 28 publications

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“…Additional selection for transposition of lox sites is not necessary in this first round because the P1 headful packaging itself serves as the selection for the low frequency of lox-site transpositions in the previous step [see references (23,24) for detailed discussions].…”

Section: Methodology For Tn10 Transposon Retrofitting Of Bacsmentioning

confidence: 99%

“…Note that loxCre independent deletions can also be packaged by the P1 headful mechanism when there is no selection for lox site transposition per se; such deletions are also efficiently isolated (23). Therefore all our loxP and lox511 transposons have been designed to alter the size of the Not I-Not I BAC-vector DNA band that arises from a Not I enzymatic digest of the deletion clone DNA [see references (23,24,27) for discussion].…”

Section: Scoring Authentic Lox-cre Recombinations By Unique Size Of Bmentioning

confidence: 99%

“…It is important to note that second round deletions from the opposite end of insert DNA requires selecting for the transposition event in addition to the BAC DNA: because first round deletions are selected by P1 headfull packaging, all starting BACs for second round deletions would be less than ~110 kb, and hence require selecting for the low frequency of transposition [see reference (23,24) for detailed discussions]. …”

Section: Truncations From the Other End Of Insert Dna In Bacsmentioning

confidence: 99%

See 2 more Smart Citations

Functionalizing Bacterial Artificial Chromosomes with Transposons to Explore Gene Regulation

Wolf¹,

Iranloye²,

Norford³

et al. 2011

Bacterial Artificial Chromosomes

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Section: Methodology For Tn10 Transposon Retrofitting Of Bacsmentioning

confidence: 99%

Section: Scoring Authentic Lox-cre Recombinations By Unique Size Of Bmentioning

confidence: 99%

Section: Truncations From the Other End Of Insert Dna In Bacsmentioning

confidence: 99%

See 1 more Smart Citation

Functionalizing Bacterial Artificial Chromosomes with Transposons to Explore Gene Regulation

Wolf¹,

Iranloye²,

Norford³

et al. 2011

Bacterial Artificial Chromosomes

View full text Add to dashboard Cite

“…If the length of DNA (B-A-C) exceeds the headful packaging capacity of ~110 kb, then the second loxP site (indicated by the thick arrows) would not fit in the phage head and the DNA cannot circularize by loxPCre recombination after entry into cells [46]. It leads to the BAC DNA not being rescued.…”

Section: Cross-recombination Between Loxp and Lox511 Sites Does Not Occmentioning

confidence: 99%

“…Thus the transposed loxP site of one orientation produces a deletion from one end of genomic insert, while the loxP inserted in the opposite orientation simply inverts the DNA between it and the one endogenous to the BAC. The phage P1 DNA circularizes upon entry into the BAC-containing cell through Cre-recombination of its terminally redundant loxP sites [46]. It then forms a co-integrate structure with the enddeleted BAC DNA, again by recombination with Cre protein in the cell.…”

Section: Deleting Bac Ends With Loxp-tn10 Transposonsmentioning

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

Chatterjee

2014

Methods in Molecular Biology

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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

Cited by 11 publications

References 28 publications

Functionalizing Bacterial Artificial Chromosomes with Transposons to Explore Gene Regulation

Functionalizing Bacterial Artificial Chromosomes with Transposons to Explore Gene Regulation

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

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