hyperactive piggyBac transposase for mammalian applications
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 do...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2011-01, Vol.108 (4), p.1531-1536 |
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| creator | Yusa, Kosuke Zhou, Liqin Li, Meng Amy Bradley, Allan Craig, Nancy L |
| description | 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. |
| doi_str_mv | 10.1073/pnas.1008322108 |
| format | Article |
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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.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.1008322108</identifier><identifier>PMID: 21205896</identifier><language>eng</language><publisher>United States: National Academy of Sciences</publisher><subject>Alternative Splicing - genetics ; Animals ; Biological Sciences ; Cell lines ; Cells ; Cells, Cultured ; Comparative Genomic Hybridization ; Deoxyribonucleic acid ; DNA ; DNA Transposable Elements - genetics ; Embryo, Mammalian - cytology ; Embryonic Stem Cells - cytology ; Embryonic Stem Cells - metabolism ; Fibroblasts - cytology ; Fibroblasts - metabolism ; Gene expression ; Genetic mutation ; Genetic transposition ; Genome - genetics ; Genomes ; Genomics ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells - cytology ; Induced Pluripotent Stem Cells - metabolism ; Mammals ; Mice ; Models, Genetic ; Moths - genetics ; Mutagenesis ; Mutagenesis, Insertional ; Mutation ; Pluripotent stem cells ; Rodents ; Saccharomyces cerevisiae - genetics ; Saccharomyces cerevisiae Proteins - genetics ; Stem cells ; Transposases - genetics ; Transposases - metabolism ; Transposons ; Yeasts</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 2011-01, Vol.108 (4), p.1531-1536</ispartof><rights>Copyright © 1993-2008 National Academy of Sciences of the United States of America</rights><rights>Copyright National Academy of Sciences Jan 25, 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c488t-b0f1ffa816edc77b92b46cb9d7ee02c40547c47c51b05e98d30e8d8e75644edf3</citedby><cites>FETCH-LOGICAL-c488t-b0f1ffa816edc77b92b46cb9d7ee02c40547c47c51b05e98d30e8d8e75644edf3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/108/4.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/41001895$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/41001895$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,315,729,782,786,805,887,27931,27932,53798,53800,58024,58257</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21205896$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Yusa, Kosuke</creatorcontrib><creatorcontrib>Zhou, Liqin</creatorcontrib><creatorcontrib>Li, Meng Amy</creatorcontrib><creatorcontrib>Bradley, Allan</creatorcontrib><creatorcontrib>Craig, Nancy L</creatorcontrib><title>hyperactive piggyBac transposase for mammalian applications</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>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.</description><subject>Alternative Splicing - genetics</subject><subject>Animals</subject><subject>Biological Sciences</subject><subject>Cell lines</subject><subject>Cells</subject><subject>Cells, Cultured</subject><subject>Comparative Genomic Hybridization</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA Transposable Elements - genetics</subject><subject>Embryo, Mammalian - cytology</subject><subject>Embryonic Stem Cells - cytology</subject><subject>Embryonic Stem Cells - metabolism</subject><subject>Fibroblasts - cytology</subject><subject>Fibroblasts - metabolism</subject><subject>Gene expression</subject><subject>Genetic mutation</subject><subject>Genetic transposition</subject><subject>Genome - genetics</subject><subject>Genomes</subject><subject>Genomics</subject><subject>HEK293 Cells</subject><subject>Humans</subject><subject>Induced Pluripotent Stem Cells - cytology</subject><subject>Induced Pluripotent Stem Cells - metabolism</subject><subject>Mammals</subject><subject>Mice</subject><subject>Models, Genetic</subject><subject>Moths - genetics</subject><subject>Mutagenesis</subject><subject>Mutagenesis, Insertional</subject><subject>Mutation</subject><subject>Pluripotent stem cells</subject><subject>Rodents</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Saccharomyces cerevisiae Proteins - genetics</subject><subject>Stem cells</subject><subject>Transposases - genetics</subject><subject>Transposases - metabolism</subject><subject>Transposons</subject><subject>Yeasts</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkN1LwzAUxYMoOj-efVKL73U3H20SBEHFLxj4oD6HNE1nxtrUpBP235sxnQqBGzi_e87lIHSM4QIDp-O-0zH9QFBCMIgtNMIgcV4yCdtoBEB4Lhhhe2g_xhkAyELALtojmEAhZDlCl-_L3gZtBvdps95Np8sbbbIh6C72Pupos8aHrNVtq-dOd5nu-7kzenC-i4dop9HzaI--5wF6u797vX3MJ88PT7fXk9wwIYa8ggY3jRa4tLXhvJKkYqWpZM2tBWIYFIyb9ApcQWGlqClYUQvLi5IxWzf0AF2tfftF1SYP26X75qoPrtVhqbx26r_SuXc19Z-KApGc02Rw_m0Q_MfCxkHN_CJ06WYlmACcusAJGq8hE3yMwTabAAxqVbZala1-y04bp3_v2vA_7f4BVpu_dkIxhQu6yjxZA7M4-LAhWArBQhZJP1vrjfZKT4OL6u2FAKaAJWWslPQLsPeZXQ</recordid><startdate>20110125</startdate><enddate>20110125</enddate><creator>Yusa, Kosuke</creator><creator>Zhou, Liqin</creator><creator>Li, Meng Amy</creator><creator>Bradley, Allan</creator><creator>Craig, Nancy L</creator><general>National Academy of Sciences</general><general>National Acad Sciences</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>5PM</scope></search><sort><creationdate>20110125</creationdate><title>hyperactive piggyBac transposase for mammalian applications</title><author>Yusa, Kosuke ; Zhou, Liqin ; Li, Meng Amy ; Bradley, Allan ; Craig, Nancy L</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c488t-b0f1ffa816edc77b92b46cb9d7ee02c40547c47c51b05e98d30e8d8e75644edf3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Alternative Splicing - genetics</topic><topic>Animals</topic><topic>Biological Sciences</topic><topic>Cell lines</topic><topic>Cells</topic><topic>Cells, Cultured</topic><topic>Comparative Genomic Hybridization</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA Transposable Elements - genetics</topic><topic>Embryo, Mammalian - cytology</topic><topic>Embryonic Stem Cells - cytology</topic><topic>Embryonic Stem Cells - metabolism</topic><topic>Fibroblasts - cytology</topic><topic>Fibroblasts - metabolism</topic><topic>Gene expression</topic><topic>Genetic mutation</topic><topic>Genetic transposition</topic><topic>Genome - genetics</topic><topic>Genomes</topic><topic>Genomics</topic><topic>HEK293 Cells</topic><topic>Humans</topic><topic>Induced Pluripotent Stem Cells - cytology</topic><topic>Induced Pluripotent Stem Cells - metabolism</topic><topic>Mammals</topic><topic>Mice</topic><topic>Models, Genetic</topic><topic>Moths - genetics</topic><topic>Mutagenesis</topic><topic>Mutagenesis, Insertional</topic><topic>Mutation</topic><topic>Pluripotent stem cells</topic><topic>Rodents</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Saccharomyces cerevisiae Proteins - genetics</topic><topic>Stem cells</topic><topic>Transposases - genetics</topic><topic>Transposases - metabolism</topic><topic>Transposons</topic><topic>Yeasts</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Yusa, Kosuke</creatorcontrib><creatorcontrib>Zhou, Liqin</creatorcontrib><creatorcontrib>Li, Meng Amy</creatorcontrib><creatorcontrib>Bradley, Allan</creatorcontrib><creatorcontrib>Craig, Nancy L</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Oncogenes and Growth Factors Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Yusa, Kosuke</au><au>Zhou, Liqin</au><au>Li, Meng Amy</au><au>Bradley, Allan</au><au>Craig, Nancy L</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>hyperactive piggyBac transposase for mammalian applications</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>2011-01-25</date><risdate>2011</risdate><volume>108</volume><issue>4</issue><spage>1531</spage><epage>1536</epage><pages>1531-1536</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>DNA transposons have been widely used for transgenesis and insertional mutagenesis in various organisms. 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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.</abstract><cop>United States</cop><pub>National Academy of Sciences</pub><pmid>21205896</pmid><doi>10.1073/pnas.1008322108</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; JSTOR Archive Collection A-Z Listing; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | Alternative Splicing - genetics Animals Biological Sciences Cell lines Cells Cells, Cultured Comparative Genomic Hybridization Deoxyribonucleic acid DNA DNA Transposable Elements - genetics Embryo, Mammalian - cytology Embryonic Stem Cells - cytology Embryonic Stem Cells - metabolism Fibroblasts - cytology Fibroblasts - metabolism Gene expression Genetic mutation Genetic transposition Genome - genetics Genomes Genomics HEK293 Cells Humans Induced Pluripotent Stem Cells - cytology Induced Pluripotent Stem Cells - metabolism Mammals Mice Models, Genetic Moths - genetics Mutagenesis Mutagenesis, Insertional Mutation Pluripotent stem cells Rodents Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae Proteins - genetics Stem cells Transposases - genetics Transposases - metabolism Transposons Yeasts |
| title | hyperactive piggyBac transposase for mammalian applications |
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