Nuclear transfer of adult and genetically modified fetal cells of the rat

Centre For Early Human Development, Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia The present study examines the handling, activation, and micromanipulation of rat eggs in an attempt to produce live young using nuclear transfer (NT) of adult a...

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Veröffentlicht in:Physiological genomics 2001-04, Vol.5 (4), p.193-204
Hauptverfasser: HAYES, ERIC, GALEA, SANDRA, VERKUYLEN, AMANDA, PERA, MARTIN, MORRISON, JOHN, LACHAM-KAPLAN, ORLY, TROUNSON, ALAN
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container_end_page 204
container_issue 4
container_start_page 193
container_title Physiological genomics
container_volume 5
creator HAYES, ERIC
GALEA, SANDRA
VERKUYLEN, AMANDA
PERA, MARTIN
MORRISON, JOHN
LACHAM-KAPLAN, ORLY
TROUNSON, ALAN
description Centre For Early Human Development, Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia The present study examines the handling, activation, and micromanipulation of rat eggs in an attempt to produce live young using nuclear transfer (NT) of adult and genetically modified rat fetal cells. Mature rat eggs cultured in calcium-free medium showed reduced rates (24%) of chromosomal dispersion ("spontaneous activation" characteristic of this species) compared with eggs cultured in calcium-containing medium (47%), but failed to survive micromanipulation procedures. High rates of parthenogenetic cleavage were obtained with chemical activation using ethanol/cycloheximide (65%) compared with other standard chemical activation methods (4–28%). This type of activation was also effective in reestablishing cleavage capability (19–71%), in a time-dependent manner, of spontaneously activated eggs arrested at a second prophase-like state. At most, two of four tested micromanipulation procedures were effective in producing NT embryos capable of morula or blastocyst development (14–16%) in vivo following transfer to mouse oviducts. NT blastocysts produced from cumulus cells and transfected rat fetal fibroblasts appeared morphologically and karyotypically normal (2 n = 42). Nocodazole-assisted metaphase enucleation and piezoelectric-assisted donor cell injection produced significant and equivocal effects on survival and cleavage rates of reconstructed embryos but failed to significantly improve in vivo morula/blastocyst development rates (16–28%) compared with unassisted micromanipulation (16%). Live births have not yet been obtained from early cleavage stage embryos ( n = 269) transferred to pseudopregnant recipient rat oviducts. Improvements in reconstituted NT embryo culture and transfer are required for these methods to be an effective means of transgenic rat production. activation; transfection; embryo; oocyte; transgenic
doi_str_mv 10.1152/physiolgenomics.2001.5.4.193
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Mature rat eggs cultured in calcium-free medium showed reduced rates (24%) of chromosomal dispersion ("spontaneous activation" characteristic of this species) compared with eggs cultured in calcium-containing medium (47%), but failed to survive micromanipulation procedures. High rates of parthenogenetic cleavage were obtained with chemical activation using ethanol/cycloheximide (65%) compared with other standard chemical activation methods (4–28%). This type of activation was also effective in reestablishing cleavage capability (19–71%), in a time-dependent manner, of spontaneously activated eggs arrested at a second prophase-like state. At most, two of four tested micromanipulation procedures were effective in producing NT embryos capable of morula or blastocyst development (14–16%) in vivo following transfer to mouse oviducts. NT blastocysts produced from cumulus cells and transfected rat fetal fibroblasts appeared morphologically and karyotypically normal (2 n = 42). Nocodazole-assisted metaphase enucleation and piezoelectric-assisted donor cell injection produced significant and equivocal effects on survival and cleavage rates of reconstructed embryos but failed to significantly improve in vivo morula/blastocyst development rates (16–28%) compared with unassisted micromanipulation (16%). Live births have not yet been obtained from early cleavage stage embryos ( n = 269) transferred to pseudopregnant recipient rat oviducts. 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Mature rat eggs cultured in calcium-free medium showed reduced rates (24%) of chromosomal dispersion ("spontaneous activation" characteristic of this species) compared with eggs cultured in calcium-containing medium (47%), but failed to survive micromanipulation procedures. High rates of parthenogenetic cleavage were obtained with chemical activation using ethanol/cycloheximide (65%) compared with other standard chemical activation methods (4–28%). This type of activation was also effective in reestablishing cleavage capability (19–71%), in a time-dependent manner, of spontaneously activated eggs arrested at a second prophase-like state. At most, two of four tested micromanipulation procedures were effective in producing NT embryos capable of morula or blastocyst development (14–16%) in vivo following transfer to mouse oviducts. NT blastocysts produced from cumulus cells and transfected rat fetal fibroblasts appeared morphologically and karyotypically normal (2 n = 42). Nocodazole-assisted metaphase enucleation and piezoelectric-assisted donor cell injection produced significant and equivocal effects on survival and cleavage rates of reconstructed embryos but failed to significantly improve in vivo morula/blastocyst development rates (16–28%) compared with unassisted micromanipulation (16%). Live births have not yet been obtained from early cleavage stage embryos ( n = 269) transferred to pseudopregnant recipient rat oviducts. 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development</subject><subject>Oocytes - ultrastructure</subject><subject>Parthenogenesis</subject><subject>Rats</subject><subject>Rats, Sprague-Dawley</subject><subject>Transfection - methods</subject><issn>1094-8341</issn><issn>1531-2267</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kEtrGzEUhUVoyMPJXyhalO5mqueMBkqhmLoJmGSTrIU8urJVNB5X0pD631fGDgVDV_cuvnvOuQehT5TUlEr2ZbfZJz-GNWzHwfepZoTQWtaiph2_QDdUclox1rQfyk46USku6DW6TelXAUWr5BW6ppQz1TXyBj0-TX0AE3GOZpscRDw6bOwUMjZbi4sLZN-bEPZ4GK13Hix2kE3APYSQDnTeAI4m36FLZ0KC-9OcodfFj5f5Q7V8_vk4_76seilprtiqZb00YN2KKykUc0K1rbAdZ7wFasmqhBRKNUTxXlJT3lGNso210IDrJJ-hz0fdXRx_T5CyHnw6hDFbGKekW6JI2zFRwK9HsI9jShGc3kU_mLjXlOhDlfqsSn2oUkstdKmynH88-UyrAey_41N3Bfh2BDZ-vXnzEd71xvVeL6YQXuBPPvc4qeuddUWA_V_gPNt7rL86pJ-_</recordid><startdate>20010427</startdate><enddate>20010427</enddate><creator>HAYES, ERIC</creator><creator>GALEA, SANDRA</creator><creator>VERKUYLEN, AMANDA</creator><creator>PERA, MARTIN</creator><creator>MORRISON, JOHN</creator><creator>LACHAM-KAPLAN, ORLY</creator><creator>TROUNSON, ALAN</creator><general>Am Physiological Soc</general><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>7X8</scope></search><sort><creationdate>20010427</creationdate><title>Nuclear transfer of adult and genetically modified fetal cells of the rat</title><author>HAYES, ERIC ; 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development</topic><topic>Oocytes - ultrastructure</topic><topic>Parthenogenesis</topic><topic>Rats</topic><topic>Rats, Sprague-Dawley</topic><topic>Transfection - methods</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>HAYES, ERIC</creatorcontrib><creatorcontrib>GALEA, SANDRA</creatorcontrib><creatorcontrib>VERKUYLEN, AMANDA</creatorcontrib><creatorcontrib>PERA, MARTIN</creatorcontrib><creatorcontrib>MORRISON, JOHN</creatorcontrib><creatorcontrib>LACHAM-KAPLAN, ORLY</creatorcontrib><creatorcontrib>TROUNSON, ALAN</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Physiological genomics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>HAYES, ERIC</au><au>GALEA, SANDRA</au><au>VERKUYLEN, AMANDA</au><au>PERA, MARTIN</au><au>MORRISON, JOHN</au><au>LACHAM-KAPLAN, ORLY</au><au>TROUNSON, ALAN</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Nuclear transfer of adult and genetically modified fetal cells of the rat</atitle><jtitle>Physiological genomics</jtitle><addtitle>Physiol Genomics</addtitle><date>2001-04-27</date><risdate>2001</risdate><volume>5</volume><issue>4</issue><spage>193</spage><epage>204</epage><pages>193-204</pages><issn>1094-8341</issn><eissn>1531-2267</eissn><abstract>Centre For Early Human Development, Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia The present study examines the handling, activation, and micromanipulation of rat eggs in an attempt to produce live young using nuclear transfer (NT) of adult and genetically modified rat fetal cells. Mature rat eggs cultured in calcium-free medium showed reduced rates (24%) of chromosomal dispersion ("spontaneous activation" characteristic of this species) compared with eggs cultured in calcium-containing medium (47%), but failed to survive micromanipulation procedures. High rates of parthenogenetic cleavage were obtained with chemical activation using ethanol/cycloheximide (65%) compared with other standard chemical activation methods (4–28%). This type of activation was also effective in reestablishing cleavage capability (19–71%), in a time-dependent manner, of spontaneously activated eggs arrested at a second prophase-like state. At most, two of four tested micromanipulation procedures were effective in producing NT embryos capable of morula or blastocyst development (14–16%) in vivo following transfer to mouse oviducts. NT blastocysts produced from cumulus cells and transfected rat fetal fibroblasts appeared morphologically and karyotypically normal (2 n = 42). Nocodazole-assisted metaphase enucleation and piezoelectric-assisted donor cell injection produced significant and equivocal effects on survival and cleavage rates of reconstructed embryos but failed to significantly improve in vivo morula/blastocyst development rates (16–28%) compared with unassisted micromanipulation (16%). Live births have not yet been obtained from early cleavage stage embryos ( n = 269) transferred to pseudopregnant recipient rat oviducts. Improvements in reconstituted NT embryo culture and transfer are required for these methods to be an effective means of transgenic rat production. activation; transfection; embryo; oocyte; transgenic</abstract><cop>United States</cop><pub>Am Physiological Soc</pub><pmid>11328965</pmid><doi>10.1152/physiolgenomics.2001.5.4.193</doi><tpages>12</tpages></addata></record>
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source MEDLINE; American Physiological Society; EZB-FREE-00999 freely available EZB journals
subjects Animals
Animals, Genetically Modified - embryology
beta-Galactosidase - genetics
Cell Culture Techniques - methods
Cell Division
Cells, Cultured
Diploidy
Embryo, Mammalian - cytology
Embryo, Mammalian - physiology
Embryonic and Fetal Development
Female
Fetus - cytology
Fibroblasts - transplantation
Green Fluorescent Proteins
Luminescent Proteins - genetics
Metaphase
Mice
Nuclear Transfer Techniques
Oocytes - cytology
Oocytes - growth & development
Oocytes - ultrastructure
Parthenogenesis
Rats
Rats, Sprague-Dawley
Transfection - methods
title Nuclear transfer of adult and genetically modified fetal cells of the rat
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