| A hyperactive piggyBac transposase for mammalian applications. | |
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MedLine Citation:
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PMID: 21205896 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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DNA transposons have been widely used for transgenesis and insertional mutagenesis in various organisms. Among the transposons active in mammalian cells, the moth-derived transposon piggyBac is most promising with its highly efficient transposition, large cargo capacity, and precise repair of the donor site. Here we report the generation of a hyperactive piggyBac transposase. The active transposition of piggyBac in multiple organisms allowed us to screen a transposase mutant library in yeast for hyperactive mutants and then to test candidates in mouse ES cells. We isolated 18 hyperactive mutants in yeast, among which five were also hyperactive in mammalian cells. By combining all mutations, a total of 7 aa substitutions, into a single reading frame, we generated a unique hyperactive piggyBac transposase with 17-fold and ninefold increases in excision and integration, respectively. We showed its applicability by demonstrating an increased efficiency of generation of transgene-free mouse induced pluripotent stem cells. We also analyzed whether this hyperactive piggyBac transposase affects the genomic integrity of the host cells. The frequency of footprints left by the hyperactive piggyBac transposase was as low as WT transposase (~1%) and we found no evidence that the expression of the transposase affects genomic integrity. This hyperactive piggyBac transposase expands the utility of the piggyBac transposon for applications in mammalian genetics and gene therapy. |
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Authors:
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Kosuke Yusa; Liqin Zhou; Meng Amy Li; Allan Bradley; Nancy L Craig |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2011-01-04 |
Journal Detail:
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Title: Proceedings of the National Academy of Sciences of the United States of America Volume: 108 ISSN: 1091-6490 ISO Abbreviation: Proc. Natl. Acad. Sci. U.S.A. Publication Date: 2011 Jan |
Date Detail:
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Created Date: 2011-01-26 Completed Date: 2011-04-26 Revised Date: 2011-07-25 |
Medline Journal Info:
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Nlm Unique ID: 7505876 Medline TA: Proc Natl Acad Sci U S A Country: United States |
Other Details:
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Languages: eng Pagination: 1531-6 Citation Subset: IM |
Affiliation:
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Wellcome Trust Sanger Institute, Cambridge CB10 1SA, United Kingdom. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Alternative Splicing
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genetics Animals Cells, Cultured Comparative Genomic Hybridization DNA Transposable Elements / genetics* Embryo, Mammalian / cytology Embryonic Stem Cells / cytology, metabolism* Fibroblasts / cytology, metabolism Genome / genetics HEK293 Cells Humans Induced Pluripotent Stem Cells / cytology, metabolism Mice Models, Genetic Moths / genetics Mutagenesis, Insertional* Mutation Saccharomyces cerevisiae / genetics Saccharomyces cerevisiae Proteins / genetics Transposases / genetics, metabolism* |
| Grant Support | |
ID/Acronym/Agency:
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WT077187//Wellcome Trust; //Howard Hughes Medical Institute |
| Chemical | |
Reg. No./Substance:
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0/DNA Transposable Elements; 0/Saccharomyces cerevisiae Proteins; 0/URA3 protein, S cerevisiae; EC 2.7.7.-/Transposases |
| Comments/Corrections | |
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