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Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos.

Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We asses... Full description

Journal Title: Reproduction (Cambridge England), January 2013, Vol.145(1), pp.97-108
Main Author: Eghbalsaied, Shahin
Other Authors: Ghaedi, Kamran , Laible, Götz , Hosseini, Sayed Morteza , Forouzanfar, Mohsen , Hajian, Mehdi , Oback, Fleur , Nasr-Esfahani, Mohammad H , Oback, Björn
Format: Electronic Article Electronic Article
Language: English
Subjects:
DNA
ID: E-ISSN: 1741-7899 ; DOI: 10.1530/REP-12-0340
Link: http://search.proquest.com/docview/1273345410/?pq-origsite=primo
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title: Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos.
format: Article
creator:
  • Eghbalsaied, Shahin
  • Ghaedi, Kamran
  • Laible, Götz
  • Hosseini, Sayed Morteza
  • Forouzanfar, Mohsen
  • Hajian, Mehdi
  • Oback, Fleur
  • Nasr-Esfahani, Mohammad H
  • Oback, Björn
subjects:
  • Animals–Genetics
  • Animals, Genetically Modified–Genetics
  • Cattle–Metabolism
  • Cells, Cultured–Pharmacology
  • DNA–Drug Effects
  • Embryo, Mammalian–Metabolism
  • Female–Methods
  • Fertilization in Vitro–Metabolism
  • Gene Transfer Techniques–Methods
  • Green Fluorescent Proteins–Methods
  • In Vitro Techniques–Drug Effects
  • Insemination, Artificial–Metabolism
  • Male–Metabolism
  • Plasmids–Metabolism
  • Sperm Injections, Intracytoplasmic–Metabolism
  • Spermatozoa–Metabolism
  • Transfection–Metabolism
  • Green Fluorescent Proteins
  • DNA
ispartof: Reproduction (Cambridge, England), January 2013, Vol.145(1), pp.97-108
description: Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.
language: eng
source:
identifier: E-ISSN: 1741-7899 ; DOI: 10.1530/REP-12-0340
fulltext: fulltext
issn:
  • 17417899
  • 1741-7899
url: Link


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titleExposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos.
creatorEghbalsaied, Shahin ; Ghaedi, Kamran ; Laible, Götz ; Hosseini, Sayed Morteza ; Forouzanfar, Mohsen ; Hajian, Mehdi ; Oback, Fleur ; Nasr-Esfahani, Mohammad H ; Oback, Björn
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ispartofReproduction (Cambridge, England), January 2013, Vol.145(1), pp.97-108
identifierE-ISSN: 1741-7899 ; DOI: 10.1530/REP-12-0340
subjectAnimals–Genetics ; Animals, Genetically Modified–Genetics ; Cattle–Metabolism ; Cells, Cultured–Pharmacology ; DNA–Drug Effects ; Embryo, Mammalian–Metabolism ; Female–Methods ; Fertilization in Vitro–Metabolism ; Gene Transfer Techniques–Methods ; Green Fluorescent Proteins–Methods ; In Vitro Techniques–Drug Effects ; Insemination, Artificial–Metabolism ; Male–Metabolism ; Plasmids–Metabolism ; Sperm Injections, Intracytoplasmic–Metabolism ; Spermatozoa–Metabolism ; Transfection–Metabolism ; Green Fluorescent Proteins ; DNA
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descriptionTransgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.
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titleExposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos.
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titleExposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos.
authorEghbalsaied, Shahin ; Ghaedi, Kamran ; Laible, Götz ; Hosseini, Sayed Morteza ; Forouzanfar, Mohsen ; Hajian, Mehdi ; Oback, Fleur ; Nasr-Esfahani, Mohammad H ; Oback, Björn
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