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Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos

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Abstract

Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19–20 h after hCG injection. About 5 μg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3′ flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 μg/mL rhFVIII in milk, with an activity of 0.594 lU/mL. The DM founder female produced 118 μg/mL rhFVIII, with activity values of 18 lU/mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.

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Authors and Affiliations

  1. Research Institute of Animal Production, Hlohovská 2, 949 92, Nitra, Slovak Republic

    Peter Chrenek, Dusan Vasicek, Alexander V. Makarevich, Rastislav Jurcik, Karin Suvegova, Vladimir Parkanyi, Miroslav Bauer & Jan Rafay

  2. National Hemophilia Center, Department of Hematology, Universtiy Hospital, Antolska 11, 851 07, Bratislava, Slovak Republic

    Angelika Batorova

  3. American Integrated Biologics, Inc., PO Box 252, E. Woodstock, CT, 06244, USA

    Rekha K. Paleyanda

Authors
  1. Peter Chrenek
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  2. Dusan Vasicek
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  3. Alexander V. Makarevich
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  4. Rastislav Jurcik
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  5. Karin Suvegova
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  6. Vladimir Parkanyi
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  7. Miroslav Bauer
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  8. Jan Rafay
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  9. Angelika Batorova
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  10. Rekha K. Paleyanda
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Correspondence to Rekha K. Paleyanda.

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Chrenek, P., Vasicek, D., Makarevich, A.V. et al. Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos. Transgenic Res 14, 417–428 (2005). https://doi.org/10.1007/s11248-005-3238-8

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  • DOI: https://doi.org/10.1007/s11248-005-3238-8

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