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Nuclear transfer of M-phase ferret fibroblasts synchronized with the microtubule inhibitor demecolcine
The development of reconstructed embryos following nuclear transfer (NT) appears to be dependent upon a variety of factors, including cell cycle synchronization between the donor nucleus and recipient oocyte. Here we use the microtubule inhibitor, demecolcine, to synchronize ferret fibroblasts in me...
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| Published in: | Journal of experimental zoology. Part A, Comparative experimental biology Comparative experimental biology, 2005-12, Vol.303A (12), p.1126-1134 |
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| cites | cdi_FETCH-LOGICAL-c3904-af446a21d0f4feed6d79905155cc1e867af2c467f9a4f56f4b2cd20e712de9dc3 |
| container_end_page | 1134 |
| container_issue | 12 |
| container_start_page | 1126 |
| container_title | Journal of experimental zoology. Part A, Comparative experimental biology |
| container_volume | 303A |
| creator | Li, Ziyi Chen, Xin Sun, Xingshen Zhou, Qi Chen, Juan Leno, Gregory H. Engelhardt, John F. |
| description | The development of reconstructed embryos following nuclear transfer (NT) appears to be dependent upon a variety of factors, including cell cycle synchronization between the donor nucleus and recipient oocyte. Here we use the microtubule inhibitor, demecolcine, to synchronize ferret fibroblasts in metaphase (M‐phase) in order to match their cell cycle position with that of the recipient oocyte at the time of NT. The fibroblasts were obtained from 28‐day fetuses and cultured for 1–30 days prior to NT. Fibroblast cultures were treated with 0.05 μg/ml of demecolcine for 3 hr or overnight (14–16 hr) after various times in culture to determine the optimal conditions for M‐phase synchronization. The percentage of G2/M‐phase cells in demecolcine‐treated cultures was significantly greater than that found in untreated cultures (P |
| doi_str_mv | 10.1002/jez.a.234 |
| format | article |
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Here we use the microtubule inhibitor, demecolcine, to synchronize ferret fibroblasts in metaphase (M‐phase) in order to match their cell cycle position with that of the recipient oocyte at the time of NT. The fibroblasts were obtained from 28‐day fetuses and cultured for 1–30 days prior to NT. Fibroblast cultures were treated with 0.05 μg/ml of demecolcine for 3 hr or overnight (14–16 hr) after various times in culture to determine the optimal conditions for M‐phase synchronization. The percentage of G2/M‐phase cells in demecolcine‐treated cultures was significantly greater than that found in untreated cultures (P<0.05). Optimally synchronized M‐phase fibroblasts were collected by mitotic shake‐off and evaluated for their effectiveness in NT. M‐phase somatic cell‐derived NT embryos reconstituted by electrofusion or microinjection underwent implantation and formed fetuses at similar rates (5.4% vs. 3.4%, and 1.8% vs. 1.2%, respectively); however, no NT embryos developed to term. In summary, these data demonstrate two important points. First, demecolcine treatment effectively synchronizes ferret fibroblasts in M‐phase of the cell cycle; and second, these somatic cells are capable of driving embryo development following NT. Our results should facilitate the development of cloned ferrets as an animal model for human lung disease such as influenza and cystic fibrosis. J. Exp. Zool. 303A:1126–1234, 2005. © 2005 Wiley‐Liss, Inc.</description><identifier>ISSN: 1548-8969</identifier><identifier>ISSN: 1932-5223</identifier><identifier>EISSN: 1552-499X</identifier><identifier>EISSN: 1932-5231</identifier><identifier>DOI: 10.1002/jez.a.234</identifier><identifier>PMID: 16254916</identifier><language>eng</language><publisher>Hoboken: Wiley Subscription Services, Inc., A Wiley Company</publisher><subject>Analysis of Variance ; Animals ; Cell Nucleus - physiology ; Cloning, Organism - methods ; Demecolcine - pharmacology ; Embryo Transfer ; Embryonic Development - physiology ; Ferrets ; Fibroblasts - cytology ; Karyotyping ; Metaphase - drug effects ; Metaphase - physiology ; Microinjections ; Microtubules - drug effects ; Microtubules - physiology ; Mustela putorius furo ; Nuclear Transfer Techniques ; Oocytes - drug effects ; Oocytes - physiology</subject><ispartof>Journal of experimental zoology. Part A, Comparative experimental biology, 2005-12, Vol.303A (12), p.1126-1134</ispartof><rights>Copyright © 2005 Wiley‐Liss, Inc., A Wiley Company</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3904-af446a21d0f4feed6d79905155cc1e867af2c467f9a4f56f4b2cd20e712de9dc3</citedby><cites>FETCH-LOGICAL-c3904-af446a21d0f4feed6d79905155cc1e867af2c467f9a4f56f4b2cd20e712de9dc3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16254916$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Li, Ziyi</creatorcontrib><creatorcontrib>Chen, Xin</creatorcontrib><creatorcontrib>Sun, Xingshen</creatorcontrib><creatorcontrib>Zhou, Qi</creatorcontrib><creatorcontrib>Chen, Juan</creatorcontrib><creatorcontrib>Leno, Gregory H.</creatorcontrib><creatorcontrib>Engelhardt, John F.</creatorcontrib><title>Nuclear transfer of M-phase ferret fibroblasts synchronized with the microtubule inhibitor demecolcine</title><title>Journal of experimental zoology. Part A, Comparative experimental biology</title><addtitle>J. Exp. Zool</addtitle><description>The development of reconstructed embryos following nuclear transfer (NT) appears to be dependent upon a variety of factors, including cell cycle synchronization between the donor nucleus and recipient oocyte. Here we use the microtubule inhibitor, demecolcine, to synchronize ferret fibroblasts in metaphase (M‐phase) in order to match their cell cycle position with that of the recipient oocyte at the time of NT. The fibroblasts were obtained from 28‐day fetuses and cultured for 1–30 days prior to NT. Fibroblast cultures were treated with 0.05 μg/ml of demecolcine for 3 hr or overnight (14–16 hr) after various times in culture to determine the optimal conditions for M‐phase synchronization. The percentage of G2/M‐phase cells in demecolcine‐treated cultures was significantly greater than that found in untreated cultures (P<0.05). Optimally synchronized M‐phase fibroblasts were collected by mitotic shake‐off and evaluated for their effectiveness in NT. M‐phase somatic cell‐derived NT embryos reconstituted by electrofusion or microinjection underwent implantation and formed fetuses at similar rates (5.4% vs. 3.4%, and 1.8% vs. 1.2%, respectively); however, no NT embryos developed to term. In summary, these data demonstrate two important points. First, demecolcine treatment effectively synchronizes ferret fibroblasts in M‐phase of the cell cycle; and second, these somatic cells are capable of driving embryo development following NT. Our results should facilitate the development of cloned ferrets as an animal model for human lung disease such as influenza and cystic fibrosis. J. Exp. Zool. 303A:1126–1234, 2005. © 2005 Wiley‐Liss, Inc.</description><subject>Analysis of Variance</subject><subject>Animals</subject><subject>Cell Nucleus - physiology</subject><subject>Cloning, Organism - methods</subject><subject>Demecolcine - pharmacology</subject><subject>Embryo Transfer</subject><subject>Embryonic Development - physiology</subject><subject>Ferrets</subject><subject>Fibroblasts - cytology</subject><subject>Karyotyping</subject><subject>Metaphase - drug effects</subject><subject>Metaphase - physiology</subject><subject>Microinjections</subject><subject>Microtubules - drug effects</subject><subject>Microtubules - physiology</subject><subject>Mustela putorius furo</subject><subject>Nuclear Transfer Techniques</subject><subject>Oocytes - drug effects</subject><subject>Oocytes - physiology</subject><issn>1548-8969</issn><issn>1932-5223</issn><issn>1552-499X</issn><issn>1932-5231</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><recordid>eNqF0E1rFTEUBuAgiq3VhX9AshJczG2SSTKTpZZaW65VUFHchExywqTOx22Sob399U25F10VVycHnrxwXoReU7KihLDjK7hbmRWr-RN0SIVgFVfq19OHN2-rVkl1gF6kdFWoJII_RwdUMsEVlYfIXy52ABNxjmZKHiKePf5cbXqTAJc1QsY-dHHuBpNywmk72T7OU7gDh29C7nHuAY_Bxjkv3TIADlMfupDniB2MYOfBhgleomfeDAle7ecR-vHx9PvJp2r95ez85P26srUivDKec2kYdcRzD-Cka5QiotxkLYVWNsYzy2XjleFeSM87Zh0j0FDmQDlbH6G3u9xNnK8XSFmPIVkYBjPBvCQt20Y0nNf_hYy0RArJC3y3g-XClCJ4vYlhNHGrKdEP7evSvja6tF_sm33o0o3g_sl93QVUO3ATBtg-nqQvTn_vAvc-pAy3f72Jf7Rs6kbon5dn-htZf5CMfC0f7wEw5KBi</recordid><startdate>20051201</startdate><enddate>20051201</enddate><creator>Li, Ziyi</creator><creator>Chen, Xin</creator><creator>Sun, Xingshen</creator><creator>Zhou, Qi</creator><creator>Chen, Juan</creator><creator>Leno, Gregory H.</creator><creator>Engelhardt, John F.</creator><general>Wiley Subscription Services, Inc., A Wiley Company</general><scope>BSCLL</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>7SN</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>20051201</creationdate><title>Nuclear transfer of M-phase ferret fibroblasts synchronized with the microtubule inhibitor demecolcine</title><author>Li, Ziyi ; Chen, Xin ; Sun, Xingshen ; Zhou, Qi ; Chen, Juan ; Leno, Gregory H. ; Engelhardt, John F.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3904-af446a21d0f4feed6d79905155cc1e867af2c467f9a4f56f4b2cd20e712de9dc3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Analysis of Variance</topic><topic>Animals</topic><topic>Cell Nucleus - physiology</topic><topic>Cloning, Organism - methods</topic><topic>Demecolcine - pharmacology</topic><topic>Embryo Transfer</topic><topic>Embryonic Development - physiology</topic><topic>Ferrets</topic><topic>Fibroblasts - cytology</topic><topic>Karyotyping</topic><topic>Metaphase - drug effects</topic><topic>Metaphase - physiology</topic><topic>Microinjections</topic><topic>Microtubules - drug effects</topic><topic>Microtubules - physiology</topic><topic>Mustela putorius furo</topic><topic>Nuclear Transfer Techniques</topic><topic>Oocytes - drug effects</topic><topic>Oocytes - physiology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Li, Ziyi</creatorcontrib><creatorcontrib>Chen, Xin</creatorcontrib><creatorcontrib>Sun, Xingshen</creatorcontrib><creatorcontrib>Zhou, Qi</creatorcontrib><creatorcontrib>Chen, Juan</creatorcontrib><creatorcontrib>Leno, Gregory H.</creatorcontrib><creatorcontrib>Engelhardt, John F.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Ecology Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of experimental zoology. 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Zool</addtitle><date>2005-12-01</date><risdate>2005</risdate><volume>303A</volume><issue>12</issue><spage>1126</spage><epage>1134</epage><pages>1126-1134</pages><issn>1548-8969</issn><issn>1932-5223</issn><eissn>1552-499X</eissn><eissn>1932-5231</eissn><abstract>The development of reconstructed embryos following nuclear transfer (NT) appears to be dependent upon a variety of factors, including cell cycle synchronization between the donor nucleus and recipient oocyte. Here we use the microtubule inhibitor, demecolcine, to synchronize ferret fibroblasts in metaphase (M‐phase) in order to match their cell cycle position with that of the recipient oocyte at the time of NT. The fibroblasts were obtained from 28‐day fetuses and cultured for 1–30 days prior to NT. Fibroblast cultures were treated with 0.05 μg/ml of demecolcine for 3 hr or overnight (14–16 hr) after various times in culture to determine the optimal conditions for M‐phase synchronization. The percentage of G2/M‐phase cells in demecolcine‐treated cultures was significantly greater than that found in untreated cultures (P<0.05). Optimally synchronized M‐phase fibroblasts were collected by mitotic shake‐off and evaluated for their effectiveness in NT. M‐phase somatic cell‐derived NT embryos reconstituted by electrofusion or microinjection underwent implantation and formed fetuses at similar rates (5.4% vs. 3.4%, and 1.8% vs. 1.2%, respectively); however, no NT embryos developed to term. In summary, these data demonstrate two important points. First, demecolcine treatment effectively synchronizes ferret fibroblasts in M‐phase of the cell cycle; and second, these somatic cells are capable of driving embryo development following NT. Our results should facilitate the development of cloned ferrets as an animal model for human lung disease such as influenza and cystic fibrosis. J. Exp. Zool. 303A:1126–1234, 2005. © 2005 Wiley‐Liss, Inc.</abstract><cop>Hoboken</cop><pub>Wiley Subscription Services, Inc., A Wiley Company</pub><pmid>16254916</pmid><doi>10.1002/jez.a.234</doi><tpages>9</tpages></addata></record> |
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| ispartof | Journal of experimental zoology. Part A, Comparative experimental biology, 2005-12, Vol.303A (12), p.1126-1134 |
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| language | eng |
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| source | Wiley-Blackwell Read & Publish Collection |
| subjects | Analysis of Variance Animals Cell Nucleus - physiology Cloning, Organism - methods Demecolcine - pharmacology Embryo Transfer Embryonic Development - physiology Ferrets Fibroblasts - cytology Karyotyping Metaphase - drug effects Metaphase - physiology Microinjections Microtubules - drug effects Microtubules - physiology Mustela putorius furo Nuclear Transfer Techniques Oocytes - drug effects Oocytes - physiology |
| title | Nuclear transfer of M-phase ferret fibroblasts synchronized with the microtubule inhibitor demecolcine |
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