Document Detail

A hyperactive piggyBac transposase for mammalian applications.
MedLine Citation:
PMID:  21205896     Owner:  NLM     Status:  MEDLINE    
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.
Kosuke Yusa; Liqin Zhou; Meng Amy Li; Allan Bradley; Nancy L Craig
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2011-01-04
Journal Detail:
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:
Created Date:  2011-01-26     Completed Date:  2011-04-26     Revised Date:  2013-07-03    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1531-6     Citation Subset:  IM    
Wellcome Trust Sanger Institute, Cambridge CB10 1SA, United Kingdom.
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MeSH Terms
Alternative Splicing / genetics
Cells, Cultured
Comparative Genomic Hybridization
DNA Transposable Elements / genetics*
Embryo, Mammalian / cytology
Embryonic Stem Cells / cytology,  metabolism*
Fibroblasts / cytology,  metabolism
Genome / genetics
HEK293 Cells
Induced Pluripotent Stem Cells / cytology,  metabolism
Models, Genetic
Moths / genetics
Mutagenesis, Insertional*
Saccharomyces cerevisiae / genetics
Saccharomyces cerevisiae Proteins / genetics
Transposases / genetics,  metabolism*
Grant Support
WT077187//Wellcome Trust; //Howard Hughes Medical Institute
Reg. No./Substance:
0/DNA Transposable Elements; 0/Saccharomyces cerevisiae Proteins; 0/URA3 protein, S cerevisiae; EC 2.7.7.-/Transposases

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