A Nucleolus-Predominant piggyBac Transposase, NP-mPB, Mediates Elevated Transposition Efficiency in Mammalian Cells

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From: PLoS ONE(Vol. 9, Issue 2)
Publisher: Public Library of Science
Document Type: Article
Length: 8,736 words
Lexile Measure: 1380L

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Author(s): Jin-Bon Hong 1,2, Fu-Ju Chou 1, Amy T. Ku 1, Hsiang-Hsuan Fan 1, Tung-Lung Lee 2, Yung-Hsin Huang 2, Tsung-Lin Yang 3,4, I-Chang Su 5, I-Shing Yu 6,7, Shu-Wha Lin 8,9, Chung-Liang Chien 1,4,10,11, Hong-Nerng Ho 1,4,10,12, You-Tzung Chen 1,4,5,10,13,*

Introduction

First cloned from the cabbage looper moth Trichoplusia ni , piggyBac is a class II DNA transposon that mobilizes DNA segments in a "cut-and paste" manner [1]. The piggyBac transposase (PBase) system has been widely applied as a genomic manipulation tool to various mammalian cell lines and model organisms, such as plants, cattle, pig, mouse, rat, rabbit, chicken, worms, fly, mosquito, planarian, yeast, protists, and several non-model insects [2]-[23]. Major features of the piggyBac system include a high transposition efficiency in different species, large cargo size, seamless removal, and relatively low insertion site preference (other than the conserved TTAA integration sequence) [3], [19], [24]-[26]. Owing to these features, the system has been used in many functional genomics studies, with particular utility for genes that are difficult to reach by other types of insertional mutagenesis vectors (e.g. , retroviruses and other transposons). Recent mutagenetic studies with the piggyBac system have been performed in mammalian gametes, embryonic stem (ES) cells, somatic cells, and cancer cell lines [7], [27]-[41].

The piggyBac system is also a candidate tool for regenerative medicine applications [42]-[44]. For induced pluripotent stem cell research, piggyBac can carry reprograming factors that enter and exit the genome without changing any nucleotides [45]-[48]. The piggyBac system has been applied to in vitro gene correction research designs in stem cells, to aid in the complete removal of a piggyBac inverted terminal repeat (ITR)-flanked drug selectable marker sequence from an exon without changing an encoded amino acid after genomic manipulations [49].

The transpositional function of mammalian codon-optimized PBase (mPB) can be maintained after mPB is fused with other proteins [34], [50]. For example, Cadinanos and Bradley fused PBase with a mutant estrogen receptor variant. Through this fusion, PBase was able to access the nucleus and mediate transposition, but only upon treatment with a steroid compound (tamoxifen) [50]. In another study, the AAV Rep-PBase fusion protein exhibited enriched capability for transposon insertion at Rep recognition sequences in the human genome [51]. Wilson et al. fused a site-specific synthetic zinc-finger DNA-binding domain (ZNF) to the N-terminus of mPB . The chimeric ZNF-mPB transposase exhibited a higher rate of site-directed genomic integration than the native mPB [52], [53]. Owens et al. fused the Gal4 DNA-binding domain (DBD) to mPB, and the chimeric Gal4-mPB facilitated transposon integration near artificially introduced upstream activating sequences [54].Transcription activator-like effector (TALE) is a new DNA-binding protein derived from the Xanthomonas sp [55], [56]. TALEs contain 34-amino acid tandem-repeat modules, which can be rearranged to target new DNA sequences [57], [58]. A TALE-linked PBase was demonstrated to direct transposition to a target region [59]. Protein function can be executed more efficiently by targeting the protein to a specific subcellular compartment. For example, a nuclear localization signal is used to direct recombinases and transcription factors to...

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Gale Document Number: GALE|A478794166