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Transgenic Research

, Volume 15, Issue 4, pp 447–454 | Cite as

Evaluation of laser-assisted lentiviral transgenesis in bovine

  • Sonja Ewerling
  • Andreas Hofmann
  • Regina Klose
  • Myriam Weppert
  • Gottfried Brem
  • Klaus Rink
  • Alexander Pfeifer
  • Eckhard WolfEmail author
Original Paper

Abstract

Lentiviral transduction of oocytes or early embryos is an efficient strategy to generate transgenic rodents and livestock. We evaluated laser-based microdrilling (MD) of the zona pellucida, which is a physical barrier for viral infection, and subsequent incubation in virus suspension as a new route for lentiviral transgenesis in bovine. Lentiviral vectors carrying an eGFP expression cassette were used to transduce oocytes or zygotes after MD as compared to the established subzonal virus injection technique (MI). The type of manipulation (MD vs. MI) did not affect cleavage rates, but had a significant effect on blastocyst rates (P < 0.001). MI of virus or sham-MI (buffer) resulted in higher blastocyst rates as compared to MD, both in the oocyte and zygote treatment groups. The latter exhibited higher rates of early cleavage (P < 0.05) and blastocyst rates (P < 0.01). The proportion of eGFP expressing blastocysts was higher after infection of oocytes (MD: 44 ± 9%; MI: 67 ± 8%) than after infection of zygotes (MD: 26 ± 8%; MI: 26 ± 9%). Overall efficacy (eGFP-positive blastocysts per treated oocytes or zygotes) was highest after MI of oocytes (18 ± 2%). Our study demonstrates the feasibility of laser-assisted lentiviral gene transfer into bovine oocytes and zygotes. However, further optimization of the procedure is required, mainly to reduce the incidence of polyspermy after MD of oocytes and to eliminate negative effects of MD on early embryonic development.

Keywords

Embryo Bovine Laser Lentivirus Transgene 

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Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Sonja Ewerling
    • 1
    • 2
  • Andreas Hofmann
    • 4
    • 5
  • Regina Klose
    • 1
  • Myriam Weppert
    • 1
  • Gottfried Brem
    • 2
  • Klaus Rink
    • 3
  • Alexander Pfeifer
    • 4
    • 5
  • Eckhard Wolf
    • 1
    Email author
  1. 1.Institute of Molecular Animal Breeding/Gene CenterLudwig–Maximilians UniversityMunichGermany
  2. 2.apoGene GmbH & Co. KGFreisingGermany
  3. 3.OCTAX Microscience GmbHHerbornGermany
  4. 4.Department of Pharmacy, Molecular Pharmacology, Center for Drug ResearchLudwig–Maximilians UniversityMunichGermany
  5. 5.Institute of Pharmacology and ToxicologyUniversity of BonnGermany

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