title: A nucleolus-predominant piggyBac transposase, NP-mPB, mediates elevated transposition efficiency in mammalian cells.
abstract: PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3-4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells.
authors.affiliation: Graduate Institute of Medical Genomics and Proteomics, National Taiwan University College of Medicine, Taipei, Taiwan ; Department of Dermatology, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan.
authors.forename: Jin-Bon
authors.initials: JB
authors.lastname: Hong
authors.affiliation: Graduate Institute of Medical Genomics and Proteomics, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Fu-Ju
authors.initials: FJ
authors.lastname: Chou
authors.affiliation: Graduate Institute of Medical Genomics and Proteomics, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Amy T
authors.initials: AT
authors.lastname: Ku
authors.affiliation: Graduate Institute of Medical Genomics and Proteomics, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Hsiang-Hsuan
authors.initials: HH
authors.lastname: Fan
authors.affiliation: Department of Dermatology, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan.
authors.forename: Tung-Lung
authors.initials: TL
authors.lastname: Lee
authors.affiliation: Department of Dermatology, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan.
authors.forename: Yung-Hsin
authors.initials: YH
authors.lastname: Huang
authors.affiliation: Department of Otolaryngology, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan ; Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Tsung-Lin
authors.initials: TL
authors.lastname: Yang
authors.affiliation: Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: I-Chang
authors.initials: IC
authors.lastname: Su
authors.affiliation: Transgenic Mouse Model Core Facility of the National Research Program for Genomic Medicine, National Taiwan University College of Medicine, Taipei, Taiwan ; Laboratory Animal Center, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: I-Shing
authors.initials: IS
authors.lastname: Yu
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan ; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Shu-Wha
authors.initials: SW
authors.lastname: Lin
authors.affiliation: Graduate Institute of Medical Genomics and Proteomics, National Taiwan University College of Medicine, Taipei, Taiwan ; Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan ; Stem Cell Core Laboratory, National Taiwan University Center of Genomic Medicine, National Taiwan University, Taipei, Taiwan ; Graduate Institute of Anatomy and Cell Biology, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Chung-Liang
authors.initials: CL
authors.lastname: Chien
authors.affiliation: Graduate Institute of Medical Genomics and Proteomics, National Taiwan University College of Medicine, Taipei, Taiwan ; Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan ; Stem Cell Core Laboratory, National Taiwan University Center of Genomic Medicine, National Taiwan University, Taipei, Taiwan ; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan.
authors.forename: Hong-Nerng
authors.initials: HN
authors.lastname: Ho
authors.affiliation: Graduate Institute of Medical Genomics and Proteomics, National Taiwan University College of Medicine, Taipei, Taiwan ; Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan ; Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan ; Stem Cell Core Laboratory, National Taiwan University Center of Genomic Medicine, National Taiwan University, Taipei, Taiwan ; Genome and Systems Biology Program, National Taiwan University, Taipei, Taiwan.
authors.forename: You-Tzung
authors.initials: YT
authors.lastname: Chen
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D003062:Codon / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D016678:Genome / Q000235:genetics; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008322:Mammals / Q000378:metabolism*; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods; D019076:Transgenes / Q000235:genetics*; D019895:Transposases / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D003062:Codon; D004251:DNA Transposable Elements; D019895:Transposases