Analysis of the piggyBac transposase reveals a functional nuclear targeting signal in the 94 c-terminal residues

Keith, James H; Fraser, Tresa S.; Fraser, Malcolm J.

doi:10.1186/1471-2199-9-72

Cited by 21 publications

(20 citation statements)

References 64 publications

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“…, 2003) are marked with an asterisk (*) above the sequences. The functional nuclear localization signal (NLS) at the C‐terminal portion of the piggyBac Tpase between amino acids 551 and 571 (dark grey background) was identified by Keith et al. (2008b) and was not found in the IDT Tpase amino acid sequence.…”

Section: Resultsmentioning

confidence: 99%

“…(2008a) have recently shown that mutation of the conserved three aspartic acids and another highly conserved aspartic acid residue (D450) close to the third proposed aspartic acid (D447) are necessary for piggyBac Tpase activity. Keith et al. (2008b) have identified a functional nuclear localization signal at the C‐terminal portion of the piggyBac Tpase between amino acids 551 and 571 which was not found in the IDT Tpase amino acid sequence.…”

Section: Discussionmentioning

confidence: 99%

“…The conserved aspartic acid residues thought to be part of a putative DDD domain of Tpases from the piggyBac superfamily (Sarkar et al, 2003) are marked with an asterisk (*) above the sequences. The functional nuclear localization signal (NLS) at the C-terminal portion of the piggyBac Tpase between amino acids 551 and 571 (dark grey background) was identified by Keith et al (2008b) and was not found in the IDT Tpase amino acid sequence. A predicted NLS (SRKRRQ) of the IDT, TN5B and SF21 Tpases are shown underlined.…”

Section: Discussionmentioning

confidence: 99%

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Molecular analysis of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) shows an interruption of an inhibitor of apoptosis gene (iap‐3) by a new class‐II piggyBac‐related insect transposon

Carpes

Nunes

Sampaio

et al. 2009

Insect Molecular Biology

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A new piggyBac-related transposable element (TE) was found in the genome of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus interrupting an inhibitor of apoptosis gene. This mutant virus induces apoptosis upon infection of an Anticarsia gemmatalis cell line, but not in a Trichoplusia ni cell line. The sequence of the new TE (which was named IDT for iap disruptor transposon) has 2531 bp with two DNA sequences flanking a putative Transposase (Tpase) ORF of 1719 bp coding for a protein with 572 amino acids. These structural features are similar to the piggyBac TE, also reported for the first time in the genome of a baculovirus. We have also isolated variants of this new TE from different lepidopteran insect cells and compared their Tpase sequences.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Molecular analysis of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) shows an interruption of an inhibitor of apoptosis gene (iap‐3) by a new class‐II piggyBac‐related insect transposon

Carpes

Nunes

Sampaio

et al. 2009

Insect Molecular Biology

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“…The transposases contain a nuclear localization signal, driving them to the nucleus [82]. An engineered PB transposase have been developed for increasing its localization within the nucleoli by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein [83].…”

Section: The Transposase Is Driven To the Nucleusmentioning

confidence: 99%

DNA Elements Tetris: A Strategy for Gene Correction

Bastie¹,

Rouleux‐Bonnin²

2016

Modern Tools for Genetic Engineering

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Transposable elements (TEs) are mobile genetic sequences that are able to move in the genome from one location to another. TEs were first regarded as junk or selfish DNA, as they comprise the largest molecular class within most metazoan genomes having no genomic function. It was necessary to wait until whole genome sequencing to provide new insights about the origin, diversity, and impact of TEs on the genome function. Thus, due to advances in molecular technology, TEs have been shown to create new regulatory sequence networks. Although nowadays most TEs present in the human genome are silenced, particularly DNA transposons, it does not mean that these sequences are dead. In this review, we detail how DNA transposons could be emphasized to create a new tool for gene correction. DNA-based transposon vectors are derived from three models: Sleeping Beauty, piggyBac, and Tol2, which all work via a "cut-and-paste" mechanism where transposase enzyme is alone able to catalyze the transposition process, which means integrating the genes of interest in chromosomal DNA. Limitations and improvements of the systems are discussed, particularly the latest way to target a specific integration site, showing that the DNA transposon-derived system and its engineering, are powerful tools for gene correction.

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“…These residues (D268, D346, D447 in piggyBac) catalyze transposition through coordination of magnesium ions. In silico predictions indicate the presence of a RING domain (blue box) in the C terminus [111,119]. The red asterisks indicate the position of the amino acid substitutions in the hyperactive transposase (PB7) [117].…”

Section: Figure 4: Schematic Of Transposon Integration Mechanismmentioning

confidence: 99%

Targeting therapeutic vector expression and integration for gene therapy applications

Burnight¹

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Gene therapy is an attractive treatment for many genetic diseases because rather than treat the symptoms of the disease, it has the potential to correct the underlying defect. Cystic fibrosis and hemophilia A are two monogenic disorders that are particularly well-suited to treatment with gene therapy as a relatively small increase in the function is needed to see improvement. Gene therapy has provided some correction in both diseases using a variety of vector systems but sustained expression and long term correction have yet to be demonstrated in the clinic. It is unclear in which cell type(s) correction of the underlying defect in cystic fibrosis will be most effective. Studies indicate that the majority of CFTR expression is in the submucosal glands and ciliated epithelia-a terminally differentiated cell type (Engelhardt, J.F. et al, 2004, Journal of Clinical Investigation). Therapeutic gene transfer would thus be most effective if achieved in a progenitor cell type. Additionally, the native regulation of CFTR has not been definitively elucidated. To this end, one goal of our studies is to develop a lentiviral vector system with heterologous promoters of varying strengths and cell specificity to aid in our selection of optimal reagents for appropriate CFTR expression. We show that use of novel internal promoters from the human PLUNC and WDR65 genes direct persistent expression in the airway. Additionally, disruption of the nasal epithelium with the detergent polidocanol eliminated reporter expression in mouse airway. Two weeks posttreatment, expression returned indicating targeting of a progenitor cell population with our novel vectors. Integrating vector systems can treat chronic diseases such as cystic fibrosis because expression can persist long term from these vectors if cells with progenitor 2 capacity are targeted (Sinn, P.L. et al, 2005, Journal of Virology). However, the potential for genotoxicity from vector-related dysregulation is a concern. Thus, a second aim of these studies was to develop a lentiviral vector that can target a specific locus in the genome. We developed a FIV vector in which the integrase was modified with a proteinbinding domain that when co-delivered with a fusion consisting of the cognate protein and a DNA binding domain would tether the vector to the appropriate locus. Unfortunately, integrase modification rendered the vector catalytically inactive. Lastly, we hoped to develop a non-viral transposon vector system (piggyBac) for gene transfer applications to the liver for treatment of hemophilia A. The recent demonstration that piggyBac transposase is highly active in mammalian cells warrants further development of this vector as an alternative to other non-viral integrating vector systems currently under investigation. We showed persistent reporter and therapeutic transgene expression in the livers of mice treated with the piggyBac vector. Furthermore, we show for the first time in vivo persistence and increased expression from the recently developed hyperactive transposase. The...

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Analysis of the piggyBac transposase reveals a functional nuclear targeting signal in the 94 c-terminal residues

Cited by 21 publications

References 64 publications

Molecular analysis of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) shows an interruption of an inhibitor of apoptosis gene (iap‐3) by a new class‐II piggyBac‐related insect transposon

Molecular analysis of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) shows an interruption of an inhibitor of apoptosis gene (iap‐3) by a new class‐II piggyBac‐related insect transposon

DNA Elements Tetris: A Strategy for Gene Correction

Targeting therapeutic vector expression and integration for gene therapy applications

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