Analysis of the cis-acting DNA elements required for piggyBac transposable element excision

Elick, Teresa A.; Lobo, Neil F.; Fraser, Malcolm J.

doi:10.1007/s004380050534

Cited by 41 publications

(37 citation statements)

References 36 publications

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“…Assays for excision and interplasmid transposition con®rmed that piggyBac transposes via a strict``cut-and-paste'' mechanism (Elick et al 1996b;Lobo et al 1999;Thibault et al 1999), inserting exclusively at 5¢-TTAA-3¢ target sites that are duplicated upon insertion (Cary et al 1989;Wang and Fraser 1993;Fraser et al 1995;Elick et al 1996b), and excising precisely (Elick et al 1996b;Fraser et al 1996) leaving no footprint. Excision assays using both wild-type and mutagenized piggyBac terminal sequences demonstrated that the element does not discriminate between proximal and distal duplicated ends, and suggest that the transposase does not ®rst recognize an internal binding site and then scan towards the ends (Elick et al 1997). These results dier from those obtained in similar experiments using the P element, for which binding to the proximal terminal repeat is highly favored (Mullins et al 1989).…”

Section: Introductionmentioning

confidence: 79%

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The minimum internal and external sequence requirements for transposition of the eukaryotic transformation vector piggyBac

Lobo

et al. 2001

Mol Gen Genomics

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The piggyBac element from Trichoplusia ni is recognized as a useful vector for transgenesis of a wide variety of species. This transposable element is 2472 bp in length, and has a complex repeat configuration consisting of an internal repeat (IR), spacer, and terminal repeat (TR) at both ends, and a single ORF encoding the transposase. Excision assays performed in microinjected T. ni embryos using plasmids deleted for progressively larger portions of the piggyBac internal sequence reveal that the 5' and 3' IR, spacer, and TR configuration is sufficient for precise excision of piggyBac when transposase is provided in trans. Interplasmid transposition assays using plasmids carrying varying lengths of intervening sequence between the piggyBac termini in T. ni demonstrate that a minimum of 55 bp of intervening sequence is required for optimal transposition, while lengths less than 40 bp result in a dramatic decrease in transposition frequency. These results suggest that the piggyBac transposase may bind both termini simultaneously before cleavage can occur, and/or that the formation of a transposition complex requires DNA bending between the two termini. Based on these results we constructed a 702-bp cartridge with minimal piggyBac 5' and 3' terminal regions separated by an intervening sequence of optimal length. Interplasmid transposition assays demonstrate that the minimal terminal configuration is sufficient to mediate transposition, and also verify that simply inserting this cartridge into an existing plasmid converts that plasmid into a non-autonomous piggyBac transposon. We also constructed a minimal piggyBac vector, pXL-Bac, that contains an internal multiple cloning site sequence between the minimal terminal regions. These vectors should greatly facilitate the utilization of the piggyBac transposon in a wide range of hosts.

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Section: Introductionmentioning

confidence: 79%

“…These results dier from those obtained in similar experiments using the P element, for which binding to the proximal terminal repeat is highly favored (Mullins et al 1989). Mutagenesis of the terminal trinucleotides or the terminal-proximate 3 bp of the TTAA target sequence eliminates excision at the altered terminus (Elick et al 1997). …”

Section: Introductionmentioning

confidence: 98%

The minimum internal and external sequence requirements for transposition of the eukaryotic transformation vector piggyBac

Lobo

et al. 2001

Mol Gen Genomics

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“…This element has been effectively harnessed, with minimized sequences identified [86]. piggyBac is active in many vertebrates tested to date [87][88][89][90].…”

Section: Pif/harbinger Familymentioning

confidence: 99%

Transposon tools hopping in vertebrates

Ni¹,

Clark²,

Fahrenkrug³

et al. 2008

Briefings in Functional Genomics and Proteomics

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In the past decade, tools derived from DNA transposons have made major contributions to vertebrate genetic studies from gene delivery to gene discovery. Multiple, highly complementary systems have been developed, and many more are in the pipeline. Judging which DNA transposon element will work the best in diverse uses from zebrafish genetic manipulation to human gene therapy is currently a complex task. We have summarized the major transposon vector systems active in vertebrates, comparing and contrasting known critical biochemical and in vivo properties, for future tool design and new genetic applications.

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“…The PB transposon, derived from the cabbage looper moth Trichoplusia ni, was originally identified in the genome of baculovirus-infected insect cells, giving rise to the name piggyBac (Cary et al, 1989;Fraser et al, 1995Fraser et al, , 1996. The original PB element was approximately 2.4 kb with identical 13-base pair (bp) terminal inverted repeats and additional asymmetric 19-bp internal repeats (Elick et al, 1997;Li et al, 2001Li et al, , 2005. PB is typically thought to mediate precise excision of transposon segments in mouse (Ding et al, 2005) and human cells through a cut-and-paste mechanism, resulting in complementary TTAA overhangs on the ends of the donor DNA and ligation of these ends to restore the donor site to its pretransposon sequence (Cary et al, 1989;Ding et al, 2005;Fraser et al, 1995;Wu et al, 2006;Wilson et al, 2007;Mitra et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

piggyBac Transposon/Transposase System to Generate CD19-Specific T Cells for the Treatment of B-Lineage Malignancies

et al. 2010

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Nonviral integrating vectors can be used for expression of therapeutic genes. piggyBac (PB), a transposon= transposase system, has been used to efficiently generate induced pluripotent stems cells from somatic cells, without genetic alteration. In this paper, we apply PB transposition to express a chimeric antigen receptor (CAR) in primary human T cells. We demonstrate that T cells electroporated to introduce the PB transposon and transposase stably express CD19-specific CAR and when cultured on CD19 þ artificial antigen-presenting cells, numerically expand in a CAR-dependent manner, display a phenotype associated with both memory and effector T cell populations, and exhibit CD19-dependent killing of tumor targets. Integration of the PB transposon expressing CAR was not associated with genotoxicity, based on chromosome analysis. PB transposition for generating human T cells with redirected specificity to a desired target such as CD19 is a new genetic approach with therapeutic implications.

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Analysis of the cis-acting DNA elements required for piggyBac transposable element excision

Cited by 41 publications

References 36 publications

The minimum internal and external sequence requirements for transposition of the eukaryotic transformation vector piggyBac

The minimum internal and external sequence requirements for transposition of the eukaryotic transformation vector piggyBac

Transposon tools hopping in vertebrates

piggyBac Transposon/Transposase System to Generate CD19-Specific T Cells for the Treatment of B-Lineage Malignancies

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