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Knocking-in of the Human Thrombopoietin Gene on Beta-casein Locus in Bovine Fibroblasts

  • Chang, Mira (Center for Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Lee, Jeong-Woong (Center for Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Koo, Deog-Bon (Department of Biotechnology, Daegu University) ;
  • Shin, Sang Tae (College of Veterinary Medicine, Chungnam National University) ;
  • Han, Yong-Mahn (Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST))
  • Received : 2009.09.28
  • Accepted : 2010.01.07
  • Published : 2010.06.01

Abstract

Animal bioreactors have been regarded as alternative tools for the production of limited human therapeutic proteins. The mammary glands of cattle are optimal tissues to produce therapeutic proteins that cannot be produced in large amounts in traditional systems based on microorganisms and eukaryotic cells. In this study, two knock-in vectors, pBCTPOKI-6 and pBCTPOKI-10, which target the hTPO gene on the bovine beta-casein locus, were designed to develop cloned transgenic cattle. The pBCTPOKI-6 and pBCTPOKI-10 vectors expressed hTPO protein in culture medium at a concentration of 774 pg/ml and 1,867 pg/ml, respectively. Successfully, two targeted cell clones were obtained from the bovine fibroblasts transfected with the pBCTPOKI-6 vector. Cloned embryos reconstructed with the targeted nuclei showed a lower in vitro developmental competence than those with the wild-type nuclei. After transfer of the cloned embryos into recipients, 7 pregnancies were detected at 40 to 60 days of gestation, but failed to develop to term. The results are the first trial for targeting of a human gene on the bovine milk protein gene locus, providing the potential for a large-scale production of therapeutic proteins in the animal bioreactor system.

Keywords

Animal Bioreactor;Bovine Beta-casein Gene;Knock-in;Human Thrombopoietin (hTPO) Gene;Bovine Fibroblasts;Somatic Cell Nuclear Transfer (SCNT))

Acknowledgement

Supported by : RDA

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