한국가축번식학회지 (Korean Journal of Animal Reproduction)

한국동물번식학회 (The Korean Society of Animal Reproduction)
권호 표지

형질전환 체세포로부터 소 복제수정란의 효율적인 생산

Efficient Production of Cloned Bovine Embryos from Transformed Somatic Cells

  • Wee G. (Laboratory of Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • B. H Sohn (Laboratory of Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Park, J. S. (Laboratory of Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • D. B. Koo (Laboratory of Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Lee, K. K. (Laboratory of Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Y. M. Han (Laboratory of Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology (KRIBB))
  • 발행 : 2003.03.01
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초록

인체 트롬보포이에틴(hTPO)은 megakaryopoiesis 과정에 주요한 역할을 하는 사이토카인이다. 따라서 이러한 트롬보포이에틴을 유선조직에서 직접적으로 발현시키기 위하여 소 베타 카제인 프로모터, 인체 트롬보포이에틴 cDNA 및 네오유전자로 구성된 발현벡터를 구축하였다. 소 귀조직 세포로부터 유도된 섬유아세포에 lipoffctamine을 이용하여 발현벡터(pBT-L n대)의 삽입을 유도하였다. G4l8 저항성을 지닌 세포의 콜로니 형성을 유도하기 위하여 2주 이상 배양을 실시하였다. 형질전환 콜로니는 PCR에 의해 동정하였으며, 이들 콜로니를 핵치환 전까지 계속적으로 증식을 유도하였다. 형질전환 세포에 의해 재구성된 난자는 전기적인 융합과 calcium ionophore와 6-DMAP를 이용한 활성화를 실시하였으며, 체외에서 7일간 배양을 실시하였다. 총 35개의 콜로니를 PCR에 의해 분석한 결과, 이 중 29(82.9%)개가 형질전환된 콜로니였다. 형질전환된 세포로 재구성된 난자의 난할율 및 배반포로의 발달율은 65.1%와 23.8%로 나타났다. 형질전환된 세포로 재구성된 난자로부터 발달한 29개의 배반포 중 27개가 형질전환으로 확인되었다. 따라서 이러한 결과들은 형질전환 소 수정란을 형질전환된 세포를 이용한 체세포 복제 기법을 통해 효과적으로 생산할 수 있다는 것을 제시하고있다.

Human thrombopoietin (hTPO) is a cytokine that plays a central role in megakaryopoiesis. To direct hTPO expression in the mammary gland, an expression vector was constructed by combining the promoter of bovine beta-casein gene, cDNA of hTPO and neomycin resistance gene (pBT-L neo). Fibroblast cells derived from cow's ear skin tissue were transfected with the expression vector (pBT-L neo) using Lipofectamine. Transfected cells resistant to G418 trea？nt were cultured to form the colonies for more than 2 weeks. The transformed colonies identified by PCR were further expanded prior to nuclear transfer. Reconstructed oocytes with transformed cells were electrofused, activated using calcium ionophore and 6-DMAP, and cultured in vitro for 7 days. Of 35 cell colonies analyzed by PCR, 29 colonies (82.9%) were positive for the hTPO gene. Cleavage and developmental rates to the blastocyst stage of reconstructed embryos with the transformed cells were 65.1% and 23.8%, respectively Of 29 blastocysts that developed from reconstructed embryos with the transformed cells, 27 embryos (93.1%) were transgenic. These results indicate that transgenic bovine embryos can be efficiently produced by somatic cell nuclear transfer using transformed cells.

키워드

참고문헌

  1. Archibald, A. L., Mcclenaghan, M., Hornsey, V., Simons, J. P. and Clark, A. J. 1990. High-level expression of biologically active human al-antitrypsin in the milk of transgenic mice. Proc. Natl. Acad. Sci. USA., 87:5178-5182 https://doi.org/10.1073/pnas.87.13.5178
  2. Bartley, T. D., Bogenberger, J., Hunt, P., Li, Y. S., Lu, H. S., Martin, F., Chang, M. S., Samal, B., Nichol, J. L., Swift, Y. S., Parker, V. P., Suggs, S., Skrine, J. D., Merewether, J. A., Clogston, C., Hsu, E., Hokom, M. M., Hornkohl, A., Choi, E., Pangelinam, M., Sun, Y., Mar, V., Mcninch, J., Simonet, L., Jacobsen, F., Xie, C., Shutter, J., Chute, H., Basu, R., Selander, L., Trollinger, D., Sieu, L.,Padilla, D., Trail, G., Elliott, G., Izumi, R., Covey, T., Crouse, J., Garcia, A., Xu, W., Castillo, J., Biron, J., Cole, S., Hu, M. C.-T., Pacifici, R., Ponting, I., Saris, C., Wen, D.,Yung, Y. P., Lin, H. and Bosselman, R. A. 1994. Identification and cloning of a mekaryocyte growth and development factor that is a ligand for the cytokine receptor. Mpl. Cell, 77:1117-1124
  3. Betthauser, J., Forsburg, E., Augenstein, M., Childs, L., Eilertsen, K., Enos, J., Forsythe, T., Goluke, P., Jurgella, G., Koppang, R., Lesmeister, T., Mallon, K., Mell, G., Misica, P., Pace, M., Pfister-Genskow, M., Strelchenko, N., Voelker, G., Watt, S., Thompson, S. and Bishop, M. 2000. Production of cloned pigs from in vitro systems. Nat. Biotechnol., 18:1055-1059 https://doi.org/10.1038/80242
  4. Bondioli, K., Ramsoondar, J., Williams, B., Costa, C. and Fodor, W. 2001. Cloned pigs generated from cultured skin fibroblasts derived from a H-transferase transgenic boar. Mol. Reprod. Dev., 60:189-195 https://doi.org/10.1002/mrd.1076
  5. Chen, R. H., Naficy, S., Logan, J. S., Diamond, L. E. and Adams, D. H. 1999. Hearts from transgenic pigs constructed with CD59/DAF genomic clones demonstrate improved survival in primates. Xeno., 6:194-200
  6. Chen, S. H., Vaught, T. D., Monahan, J. A., Boone, J., Emslie, E., Jobst, P. M., Lamborn, A. E., Schnieke, A., Robertson, L., Colman, A., Dai, Y., Polejaeva, I. A. and Ayares, D. L. 2002. Efficient production of transgenic cloned calves using preimplantation screening. BioI. Reprod. 67:1488-1492 https://doi.org/10.1095/biolreprod.102.006981
  7. Cibelli, J. B., Stice, S. L., Golueke, P. J., Kane, J. J., Jerry, J., Blackwell, C., Ponce de Leon, F. A. and Robl, J. M. 1998. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Scienece, 280:1256-1258 https://doi.org/10.1126/science.280.5367.1256
  8. Clark, A. J., Bessos, H., Bishop, J. O., Brown, P., Harris, S., Lathe, R., McClenaghan, M., Prowse, C., Simons, J. P., Whitelaw, C. B. A. and Willmut, I. 1989. Expression of human anti-hemophilic factor IV in the milk of transgenic sheep. Bio/Tech., 7:487-492 https://doi.org/10.1038/nbt0589-487
  9. Damak, S., Su, H. Y., Jay, N. P. and Bullock, D. W. 1996. Improved wool production in transgenic sheep expressing insulin-like growth factor J. Bio/Tech., 14:185-188 https://doi.org/10.1038/nbt0296-185
  10. Denning, C., Burl, S., Ainslie, A., Bracken, J., Dinnyes, A., Fletcher, J., King, T., Ritchie, M., Ritchie, W. A., Rollo, M., de Sousa, P., Travers, A., Wilmut, I. and Clark, A. I. 2001. Delection of the(1,3)galactosyl transferase(GGTAl) gene and the prion protein (PrP) gene in sheep. Nat. Biotechnol., 19:559-562 https://doi.org/10.1038/89313
  11. De Sauvage, F. J., Hass, P. E., Spencer, D. D., Malloy, B. E., Gurney, A. L., Spencer, S. A., Darbonne, W. C., Henzel, W. J., Wong, S. C., Kuang, W. J., Oles, K. J., hultgren, B., Solberg,J. R., Goeddel, D. V. and Eaton, D. 1. 1994. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature, 369: 533-538 https://doi.org/10.1038/369533a0
  12. Ebert, K. M., Selgrath, J. P., DiTullio, Denman, J., Smith, T. E., Memon, M. A., Schindler, J.E., Monastersky, G. M., Vitale, J. A. and Gordon, K. 1991. Transgenic production of a variant of human tissue-type plasminogen activator in goat milk: generation of transgenic goats. Bio/Tech., 9:835-838 https://doi.org/10.1038/nbt0991-835
  13. Echelard, Y., Destrempes, M. M., Koster, J. A., Blackwell, C., Groen, W., Pollock, D., Williams, J. L., Behboodi, E., Pommer, J. and Meade, H. M. 2002. Production of recombinant human serum albumin in the milk of transgenic cows. Theriogenology, 57:779
  14. Eyestorne, W. H. 1994. Challenges and progress in the production of transgenic cattle. Reprod Fert. Dev., 6:647-652 https://doi.org/10.1071/RD9940647
  15. Gordon, K., Lee, E., Vitale, J. A., Smith, A. E., Westphal, H. and Henninghausen, L. 1992. Production of human tissue plasminogen activator in transgenic mouse milk. Bio/Tech.,24:425-428
  16. Hammer, R. E., Pursel, V. G., Rexroad, C. E., Wall, R. J., Bolt, D. J., Ebert, K. M., Palmiter, R. D. and Brinster, R. L. 1985. Production of transgenic rabbits, sheep and pigs by microinjetion. Nature, 315:680-683 https://doi.org/10.1038/315680a0
  17. Hill, J. R., Winger, Q. A., Long, C. R., Looney, C. R., Thompson, J. A., Westhusin, M. E. 2000. Development rates of male bovine nuclear transfer embryos derived from adult and fetal cells. BioI. Reprod., 62:1135-1140 https://doi.org/10.1095/biolreprod62.5.1135
  18. Krimpenfort, P., Rademakers, A., Eyestone, W., Schans, A., Brock, S., Kooiman, P., Kootwijk, E., Platenburg, G., Pieper, F., Strijker, R. and Boer, H. 1991. Generation of transgenic dairy cattle using in vitro embryo production. Bio/Tech., 9:844-847 https://doi.org/10.1038/nbt0991-844
  19. Lai, L., Kolber-Simonds, D., Park, K. W., Cheong, H. T., Greenstein, J. L., Im, G. S., Samuel, M., Bonk, A., Rieke, A., Day, B. N., Murphy, C. N., Carter, D. B., Hawley, R. J. and Prather, R. S. 2002. Production of alpha-l,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science, 295: 1089-109 https://doi.org/10.1126/science.1068228
  20. McCreath, K. L., Howcroff, J., Campbell, K. H. S., Colman, A., Schnieke, A. E. and Kind, A. J. 2000. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature, 405:1066-1069 https://doi.org/10.1038/35016604
  21. Park, K. W., Lai, L., Cheong, H. T., Cabot, R., Sun, Q. Y., Wu, G., Rucker, E. B., Durtschi, D., Bonk, A., Samuel, M., Rieke, A., Day, B. N., Murphy, C. N., Carter, D. B. and Prather, R. S. 2002. Mosaic expression in nuclear transfer-derived embryos and the production of cloned transgenic pigs from ear-derived fibroblasts. BioI. Reprod., 66:1001-1005 https://doi.org/10.1095/biolreprod66.4.1001
  22. Parrish, J. J., Susko-Parrish, J. L., Winer, M. A. and First, N. L. 1988. Capacitation of bovine sperm by heparin. BioI. Peprod., 38:1171-1180
  23. Pintado, B. and Gutierrez-Adan, A. 1999. Transgenesis in large domestic species: future development for milk modification. Reprod. Nutr. Dev., 39:535-544 https://doi.org/10.1051/rnd:19990502
  24. Schnieke, A. E., Kind, A. J., Ritchie, W. A., Mycock, K., Scott, A. R., Ritchie, M., Willmut, I., Colman, A. and Campbell, K. H. S. 1997. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science, 278:2130-2133
  25. Sohma, Y., Akahori, H., Seki, N., Hori, T., Ogami, K., Kato, T., Shimada, Y., Kawamura, K. and Miyazaki, H. 1994. Molecular cloning and chromosomal localization of the human thrombopoietin gene. FEBS Lett., 353:57-61
  26. Sohn, B. H., Kim, S. J., Park, H., Park, S. K., Lee, C. S., Hong, H. J., Park, Y. S. and Lee, K. K. 1999. Expression and characterization of bioactive human thrombopoietin in the milk of transgenic mice. DNA Cell BioI., 18:845-852 https://doi.org/10.1089/104454999314845
  27. van Berkel, P. H. C., Welling, M. M., Geerts, M., van Veen, H. A., Ravensbergen, B., Salaheddine, M., Pauwels E. K. J., Pieper, F., Nuijens, J. H. and Nibbering, P. 2002. Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nat. Biotechnol., 20:484-487 https://doi.org/10.1038/nbt0502-484
  28. Velander, W. H., Johnson, J. L., Page, R. L., Russell, C. G., Subramanian, A., Wilkins, T. D., Gwazdauskas, F. C, Pittius, C. and Drohan, W. N. 1992a. High-level expression of a heterologous protein in the milk of transgenic swine using the cDNA encoding human protein C. Proc. Natl. Acad. Sci. USA., 89: 12003 -12007
  29. Velander, W. H., Page, R. L., Morcol, T., Russell, C. G., Cajseco, R., Young, J. M., Drohan, W. N., Gwazdauskas, F. C., Wilkins, T. D. and Hohnson, J. L. 1992b. Production of biologically active human protein C in the milk of transgenic mice. Ann. N. Y. Acad. Sci., 665:391-403
  30. Wall, R. J., Pursel, V. G., Shamay, A., McKnight, R. A., Pittius, C. W. and Hennighausen, L. 1991. High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine. Proc. Natl. Acad. Sci. USA., 88:1696-1700
  31. Zakhartchenko, V., Mueller, S., Alberio, R., Schemthaner, W., Stojkovic, M., Wenigerkind, H., Wanke, R., Lassing, C., Mueller, M., Wolf, E. and Brem, G. 2001. Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts. Mol. Reprod. Dev., 60:362-369 https://doi.org/10.1002/mrd.1098
  32. Zou, X., Wang, Y., Cheng, Y., Yang, Y., Ju, H., Tang, H., Shen, Y., Mu, A., Xu, S. and Du, M. 2002. Generation of cloned goats (Capra hircus) from transfected foetal fibroblast cells, the effect of donor cell cycle. Mol. Reprod. Dev., 61:164-172 https://doi.org/10.1002/mrd.1143