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


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.


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


Supported by : RDA


  1. Gao, X., A. Kemper and B. Popko. 1999. Advanced transgenic and gene-targeting approaches. Neurochem. Res. 24(9):1181-1188
  2. Grossmann, A., J. Lenox, H. P. Ren, J. M. Humes, J. W. Forstrom, K. Kaushansky and K. H. Sprugel. 1996. Thrombopoietin accelerates platelet, red blood cell, and neutrophil recovery in myelosuppressed mice. Exp. Hematol. 24(10):1238-1246
  3. Kim, S. J., B. H. Sohn, S. Jeong, K. W. Pak, J. S. Park, I. Y. Park, T. H. Lee, Y. H. Choi, C. S. Lee, Y. M. Han, D. Y. Yu and K. K. Lee. 1999. High-level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence. J. Biochem. (Tokyo) 126(2):320-325
  4. McCreath, K. J., J. Howcroft, K. H. Campbell, A. Colman, A. E. Schnieke and A. J. Kind. 2000. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 405(6790):1066-1069
  5. Scheerer, J. B. and G. M. Adair. 1994. Homology dependence of targeted recombination at the Chinese hamster APRT locus. Mol. Cell. Biol. 14(10):6663-6673
  6. Thomson, A. J., M. M. Marques and J. McWhir. 2003. Gene targeting in livestock. Reprod. Suppl. 61:495-508
  7. Fanucchi, M., J. Glaspy, J. Crawford, J. Garst, R. Figlin, W. Sheridan, D. Menchaca, D. Tomita, H. Ozer and L. Harker. 1997. Effects of polyethylene glycol-conjugated recombinant human megakaryocyte growth and development factor on platelet counts after chemotherapy for lung cancer. N. Engl. J. Med. 336(6):404-409
  8. Burdon, T. G., K. A. Maitland, A. J. Clark, R. Wallace and C. J. Watson. 1994. Regulation of the sheep beta-lactoglobulin gene by lactogenic hormones is mediated by a transcription factor that binds an interferon-gamma activation site-related element. Mol. Endocrinol. 8(11):1528-1536
  9. Thomas, K. R. and M. R. Capecchi. 1987. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51(3):503-512
  10. Sohn, B. H., H. G. Chang, H. S. Kang, H. Yoon, Y. S. Bae, K. K. Lee and S. J. Kim. 2003. High level expression of the bioactive human interleukin-10 in milk of transgenic mice. J. Biotechnol. 103(1):11-19
  11. Cibelli, J. B., S. L. Stice, P. J. Golueke, J. J. Kane, J. Jerry, C. Blackwell, F. A. Ponce de Leon and J. M. Robl. 1998. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280(5367):1256-1258
  12. Clark, A. J. 1998. The mammary gland as a bioreactor: expression, processing, and production of recombinant proteins. J. Mammary Gland Biol. Neoplasia 3 (3):337-350
  13. Eyestone, W. H. 1994. Challenges and progress in the production of transgenic cattle. Reprod. Fertil. Dev. 6(5):647-652
  14. Koo, D. B., Y. K. Kang, Y. H. Choi, J. S. Park, S. K. Han, I. Y. Park, S. U. Kim, K. K. Lee, D. S. Son, W. K. Chang and Y. M. Han. 2000. In vitro development of reconstructed porcine oocytes after somatic cell nuclear transfer. Biol. Reprod. 63(4):986-992
  15. Koo, D. B., Y. K. Kang, Y. H. Choi, J. S. Park, H. N. Kim, K. B. Oh, D. S. Son, H. Park, K. K. Lee and Y. M. Han. 2002. Aberrant allocations of inner cell mass and trophectoderm cells in bovine nuclear transfer blastocysts. Biol. Reprod. 67(2):487-492
  16. Piedrahita, J. A. 2000. Targeted modification of the domestic animal genome. Theriogenology 53(1):105-116
  17. Kumar, S., A. R. Clarke, M. L. Hooper, D. S. Horne, A. J. Law, J. Leaver, A. Springbett, E. Stevenson and J. P. Simons. 1994. Milk composition and lactation of beta-casein-deficient mice. Proc. Natl. Acad. Sci. USA. 91(13):6138-6142