The piggyBac transposon-mediated expression of SV40 T antigen efficiently immortalizes mouse embryonic fibroblasts (MEFs)
Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs' basic re...
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| Veröffentlicht in: | PloS one 2014-05, Vol.9 (5), p.e97316-e97316 |
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| creator | Wang, Ning Zhang, Wenwen Cui, Jing Zhang, Hongmei Chen, Xiang Li, Ruidong Wu, Ningning Chen, Xian Wen, Sheng Zhang, Junhui Yin, Liangjun Deng, Fang Liao, Zhan Zhang, Zhonglin Zhang, Qian Yan, Zhengjian Liu, Wei Ye, Jixing Deng, Youlin Wang, Zhongliang Qiao, Min Luu, Hue H Haydon, Rex C Shi, Lewis L Liang, Houjie He, Tong-Chuan |
| description | Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs' basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies. |
| doi_str_mv | 10.1371/journal.pone.0097316 |
| format | Article |
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Primary MEFs have limited life span in culture, which thus hampers MEFs' basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0097316</identifier><identifier>PMID: 24845466</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Adipose tissue ; Animals ; Antigens, Polyomavirus Transforming - biosynthesis ; Antigens, Polyomavirus Transforming - genetics ; Biocompatibility ; Biology and Life Sciences ; Biomedical materials ; Bone morphogenetic protein 9 ; Cartilage ; Cell adhesion & migration ; Cell culture ; Cell Differentiation ; Cell Line, Transformed ; Cell proliferation ; Cell self-renewal ; Cells (biology) ; Divisions ; DNA Transposable Elements ; Education ; Embryo fibroblasts ; Embryo, Mammalian ; Embryos ; Fibroblasts ; Fibroblasts - cytology ; Fibroblasts - metabolism ; FLP recombinase ; Gene Expression ; HEK293 Cells ; Humans ; Implantation ; Laboratories ; Life span ; Medicine ; Mesenchymal stem cells ; Mesenchyme ; Mice ; Multipotent Stem Cells - cytology ; Multipotent Stem Cells - metabolism ; Oncology ; Progenitor cells ; Recombinase ; Simian virus 40 ; Stem cells ; Stimulation ; Surgery ; Target recognition ; Tissues ; Tracking stock ; Translation ; Transposition ; Transposons ; Tumor antigens ; Wang, Wenwen</subject><ispartof>PloS one, 2014-05, Vol.9 (5), p.e97316-e97316</ispartof><rights>COPYRIGHT 2014 Public Library of Science</rights><rights>2014 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2014 Wang et al 2014 Wang et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-68b4d53de050713ae8365b50cf70dbac756f969ae0d4474aa210148b933771da3</citedby><cites>FETCH-LOGICAL-c692t-68b4d53de050713ae8365b50cf70dbac756f969ae0d4474aa210148b933771da3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4028212/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4028212/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24845466$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wang, Ning</creatorcontrib><creatorcontrib>Zhang, Wenwen</creatorcontrib><creatorcontrib>Cui, Jing</creatorcontrib><creatorcontrib>Zhang, Hongmei</creatorcontrib><creatorcontrib>Chen, Xiang</creatorcontrib><creatorcontrib>Li, Ruidong</creatorcontrib><creatorcontrib>Wu, Ningning</creatorcontrib><creatorcontrib>Chen, Xian</creatorcontrib><creatorcontrib>Wen, Sheng</creatorcontrib><creatorcontrib>Zhang, Junhui</creatorcontrib><creatorcontrib>Yin, Liangjun</creatorcontrib><creatorcontrib>Deng, Fang</creatorcontrib><creatorcontrib>Liao, Zhan</creatorcontrib><creatorcontrib>Zhang, Zhonglin</creatorcontrib><creatorcontrib>Zhang, Qian</creatorcontrib><creatorcontrib>Yan, Zhengjian</creatorcontrib><creatorcontrib>Liu, Wei</creatorcontrib><creatorcontrib>Ye, Jixing</creatorcontrib><creatorcontrib>Deng, Youlin</creatorcontrib><creatorcontrib>Wang, Zhongliang</creatorcontrib><creatorcontrib>Qiao, Min</creatorcontrib><creatorcontrib>Luu, Hue H</creatorcontrib><creatorcontrib>Haydon, Rex C</creatorcontrib><creatorcontrib>Shi, Lewis L</creatorcontrib><creatorcontrib>Liang, Houjie</creatorcontrib><creatorcontrib>He, Tong-Chuan</creatorcontrib><title>The piggyBac transposon-mediated expression of SV40 T antigen efficiently immortalizes mouse embryonic fibroblasts (MEFs)</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs' basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies.</description><subject>Adipose tissue</subject><subject>Animals</subject><subject>Antigens, Polyomavirus Transforming - biosynthesis</subject><subject>Antigens, Polyomavirus Transforming - genetics</subject><subject>Biocompatibility</subject><subject>Biology and Life Sciences</subject><subject>Biomedical materials</subject><subject>Bone morphogenetic protein 9</subject><subject>Cartilage</subject><subject>Cell adhesion & migration</subject><subject>Cell culture</subject><subject>Cell Differentiation</subject><subject>Cell Line, Transformed</subject><subject>Cell proliferation</subject><subject>Cell self-renewal</subject><subject>Cells (biology)</subject><subject>Divisions</subject><subject>DNA Transposable Elements</subject><subject>Education</subject><subject>Embryo fibroblasts</subject><subject>Embryo, Mammalian</subject><subject>Embryos</subject><subject>Fibroblasts</subject><subject>Fibroblasts - cytology</subject><subject>Fibroblasts - metabolism</subject><subject>FLP recombinase</subject><subject>Gene Expression</subject><subject>HEK293 Cells</subject><subject>Humans</subject><subject>Implantation</subject><subject>Laboratories</subject><subject>Life span</subject><subject>Medicine</subject><subject>Mesenchymal stem cells</subject><subject>Mesenchyme</subject><subject>Mice</subject><subject>Multipotent Stem Cells - cytology</subject><subject>Multipotent Stem Cells - metabolism</subject><subject>Oncology</subject><subject>Progenitor cells</subject><subject>Recombinase</subject><subject>Simian virus 40</subject><subject>Stem cells</subject><subject>Stimulation</subject><subject>Surgery</subject><subject>Target recognition</subject><subject>Tissues</subject><subject>Tracking stock</subject><subject>Translation</subject><subject>Transposition</subject><subject>Transposons</subject><subject>Tumor antigens</subject><subject>Wang, Wenwen</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk11rFDEUhgdRbK3-A9GAIO3FrvmazMyNUEurhUrBrr0NZzJnZlNmJtskK11_vVm7LbvSC8lFwslz3pO8ycmyt4xOmSjYpxu39CP004UbcUppVQimnmX7rBJ8ojgVz7fWe9mrEG4ozUWp1Mtsj8tS5lKp_Ww1myNZ2K5bfQFDoocxLFxw42TAxkLEhuDdwmMI1o3EteTqWlIyIzBG2-FIsG2tsTjGfkXsMDgfobe_MZDBLQMSHGq_cqM1pLW1d3UPIQZy-P30LBy9zl600Ad8s5kPsp9np7OTb5OLy6_nJ8cXE6MqHieqrGWTiwZpTgsmAEuh8jqnpi1oU4MpctVWqgKkjZSFBOCMMlnWlRBFwRoQB9n7e91F74LeuBY0y7niPOdlmYjze6JxcKMX3g7gV9qB1X8DzncafLSmR91CVSNrRSpVyVypGhoq65wXYNJgRdL6vKm2rJOFJlnjod8R3d0Z7Vx37peWlJec8SRwuBHw7naJIerBBoN9DyMmT9fnLgRXSq3RD_-gT99uQ3WQLmDH1qW6Zi2qjyUrmajyQiRq-gSVRoODNemLtTbFdxKOdhISE_EudrAMQZ9f_fh_9vJ6l_24xc4R-jgPrl_G9AHDLijvQeNdCB7bR5MZ1esOeXBDrztEbzokpb3bfqDHpIeWEH8AAtIMCg</recordid><startdate>20140520</startdate><enddate>20140520</enddate><creator>Wang, 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piggyBac transposon-mediated expression of SV40 T antigen efficiently immortalizes mouse embryonic fibroblasts (MEFs)</title><author>Wang, Ning ; Zhang, Wenwen ; Cui, Jing ; Zhang, Hongmei ; Chen, Xiang ; Li, Ruidong ; Wu, Ningning ; Chen, Xian ; Wen, Sheng ; Zhang, Junhui ; Yin, Liangjun ; Deng, Fang ; Liao, Zhan ; Zhang, Zhonglin ; Zhang, Qian ; Yan, Zhengjian ; Liu, Wei ; Ye, Jixing ; Deng, Youlin ; Wang, Zhongliang ; Qiao, Min ; Luu, Hue H ; Haydon, Rex C ; Shi, Lewis L ; Liang, Houjie ; He, Tong-Chuan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-68b4d53de050713ae8365b50cf70dbac756f969ae0d4474aa210148b933771da3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Adipose tissue</topic><topic>Animals</topic><topic>Antigens, Polyomavirus Transforming - biosynthesis</topic><topic>Antigens, Polyomavirus Transforming - genetics</topic><topic>Biocompatibility</topic><topic>Biology and Life Sciences</topic><topic>Biomedical materials</topic><topic>Bone morphogenetic protein 9</topic><topic>Cartilage</topic><topic>Cell adhesion & migration</topic><topic>Cell culture</topic><topic>Cell Differentiation</topic><topic>Cell Line, Transformed</topic><topic>Cell proliferation</topic><topic>Cell self-renewal</topic><topic>Cells (biology)</topic><topic>Divisions</topic><topic>DNA Transposable Elements</topic><topic>Education</topic><topic>Embryo fibroblasts</topic><topic>Embryo, Mammalian</topic><topic>Embryos</topic><topic>Fibroblasts</topic><topic>Fibroblasts - cytology</topic><topic>Fibroblasts - metabolism</topic><topic>FLP recombinase</topic><topic>Gene Expression</topic><topic>HEK293 Cells</topic><topic>Humans</topic><topic>Implantation</topic><topic>Laboratories</topic><topic>Life span</topic><topic>Medicine</topic><topic>Mesenchymal stem 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Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Engineering Collection</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wang, Ning</au><au>Zhang, Wenwen</au><au>Cui, Jing</au><au>Zhang, Hongmei</au><au>Chen, Xiang</au><au>Li, Ruidong</au><au>Wu, Ningning</au><au>Chen, Xian</au><au>Wen, Sheng</au><au>Zhang, Junhui</au><au>Yin, Liangjun</au><au>Deng, Fang</au><au>Liao, Zhan</au><au>Zhang, Zhonglin</au><au>Zhang, Qian</au><au>Yan, Zhengjian</au><au>Liu, Wei</au><au>Ye, Jixing</au><au>Deng, Youlin</au><au>Wang, Zhongliang</au><au>Qiao, Min</au><au>Luu, Hue H</au><au>Haydon, Rex C</au><au>Shi, Lewis L</au><au>Liang, Houjie</au><au>He, Tong-Chuan</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>The piggyBac transposon-mediated expression of SV40 T antigen efficiently immortalizes mouse embryonic fibroblasts (MEFs)</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2014-05-20</date><risdate>2014</risdate><volume>9</volume><issue>5</issue><spage>e97316</spage><epage>e97316</epage><pages>e97316-e97316</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs' basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>24845466</pmid><doi>10.1371/journal.pone.0097316</doi><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2014-05, Vol.9 (5), p.e97316-e97316 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1526225288 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Adipose tissue Animals Antigens, Polyomavirus Transforming - biosynthesis Antigens, Polyomavirus Transforming - genetics Biocompatibility Biology and Life Sciences Biomedical materials Bone morphogenetic protein 9 Cartilage Cell adhesion & migration Cell culture Cell Differentiation Cell Line, Transformed Cell proliferation Cell self-renewal Cells (biology) Divisions DNA Transposable Elements Education Embryo fibroblasts Embryo, Mammalian Embryos Fibroblasts Fibroblasts - cytology Fibroblasts - metabolism FLP recombinase Gene Expression HEK293 Cells Humans Implantation Laboratories Life span Medicine Mesenchymal stem cells Mesenchyme Mice Multipotent Stem Cells - cytology Multipotent Stem Cells - metabolism Oncology Progenitor cells Recombinase Simian virus 40 Stem cells Stimulation Surgery Target recognition Tissues Tracking stock Translation Transposition Transposons Tumor antigens Wang, Wenwen |
| title | The piggyBac transposon-mediated expression of SV40 T antigen efficiently immortalizes mouse embryonic fibroblasts (MEFs) |
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