Development of Somatic Cell Nuclear Transfer Embryos Following Donor Cell Type and Cell Treatment in Cattle

Donor 세포의 종류 및 세포처리에 따른 소 체세포 핵이식란의 체외발육

  • 손준규 (강원대학교 동물자원과학대학) ;
  • 박정준 (축산연구소) ;
  • 박춘근 (강원대학교 동물자원과학대학) ;
  • 양부근 (강원대학교 동물자원과학대학) ;
  • 김정익 (강원대학교 동물자원과학대학) ;
  • 정희태 (강원대학교 동물자원과학대학)
  • Published : 2004.03.01

Abstract

This study was conducted to investigate the effects of donor cell type, individual, passage number and trypsinization time on the in vitro development of bovine somatic cell nuclear transfer embryos. Three cell types (skin, muscle and cumulus cells) and cells from 3 individuals were used for nuclear transfer. Cell were passaged by 5, 15 or 30 times, and cell were trypsinized for 1 or 3 min before injection. Nuclear transfer were performed by conventional fusion method. Development rates to the blastocyst stage were not significantly different among three cell types (16.5∼23.9%) and individuals (16.4∼19.5%). Blastocyst formation rate of cloned embryos reconstituted with cells at passage 30 (5.8%) was significantly lower than those of embryos reconstituted with 5- and 15-passaged cells (25.3 and 23.5%, respectively, P<0.05). The rate of embryos developed to the blastocyst stage was higher in embryos reconstituted with cells trypsinized for 1 min (30.7%) compared to embryos reconstituted with cells trypsinized for 3 min (P<0.05). The result of the present study indicates that different donor cell types and individuals used in this study did not affect the development of cloned bovine embryos. However, passage number and trypsinization time of donor cells affect the in vitro development of cloned bovine embryos.

본 연구는 체세포의 종류, 세포 제공 개체, 계대배양 수 및 세포의 trypsin 처리시간이 소 체세포 핵이식란의 체외발육에 미치는 영향을 검토하였다. 세 종류의 체세포(피부, 근육 및 난구세포) 와 암소 3 개체를 실험에 공시하였으며, 한 개체 유래의 피부세포는 5∼30회 계대배양하였고, 핵이식 전에 1∼3분간 trypsin처리하여 핵이식에 사용하였다. 핵이식과정은 상법에 따라 전기융합법을 이용하였다. 핵이식란의 배반포 발육율은 세포의 종류(16.5∼23.9%)나 개체 간(16.4∼19.5%)에 차이가 없으나, 30회 계대 배양한 세포를 사용한 경우(5.8%)에는 5회(25.3%) 또는 15회(23.5%) 계대 배양한 세포를 사용한 경우에 비해 유의적으로 낮았다(P<0.05). 또한, 1분간 trypsinization 한 세포를 사용한 경우(30.7%)는 3분간 trypsinization 한 경우에 비해 배반포 발육율이 유의적으로 높게 나타났다(P<0.05). 본 연구의 결과는 소 체세포 핵이식란의 발육이 체세포의 종류 및 세포 제공 개체에 따라서는 영향을 받지 않으나, passage 수 및 체세포의 trypsinization 시간에 따라서는 영향을 받을 수 있음을 시사한다.

Keywords

References

  1. Arat, S., Rzucidlo, S. J., Gibbons, J, Miyoshi, K. and Stice, S. L. 2001. Production of transgenic bovine embryos by transfer of transfected granulosa cells into enucleated oocytes. Mol. Reprod. Dev. 60:20-26 https://doi.org/10.1002/mrd.1057
  2. Baguisi, A., Behboodi, E., Melican, D. T., Pollock, J. S., Destrempes, M. M., Cammuso, C., Williams, J. L., Nims, S. D., Porter, C. A, Midura, P., Palacios, M. J., Ayres, S. L., Denniston, R. S., Hayes, M. L., Ziomek, C. A., Meade, H. M., Godke, R. A., Gavin, W. G., Overstrom, E. W. and Echelard, Y. 1999. Production of goats by somatic cell nuclear transfer. Nature Biotech. 17:456-461 https://doi.org/10.1038/8632
  3. Betthauser, J., Forsberg, E., Augenstein, M., Childs, L., Eilertsen, K., Enos, J., Forsythe, T., Golueke, P., Jurgella, G., Koppang, R., Lesmeister, T., Mallon, K., Mell, G., Misica, P., Pace, M., Genskow, M. P., Strelchenko, N., Voelker, G., Wart, S., Thompson, S. and Bishop, M. 2000. Production of cloned pigs from in vitro systems. Nature Biotech. 18:1055-1059
  4. Brem, G. and Kuhholzer, B. 2002. The recent history of somatic cloning in mammals. Cloning Stem cells 4:57-63
  5. Campbell, K. H. S., McWhir, J., Ritchie, W. A. and Wilmut, I. 1996. Sheep cloned by nuclear transfer from a cultured cell line. Nature 380:64-66
  6. Chesne, P., Adenot, P. G., Vigilietta, C., Baratte, M., Boulanger, L. and Renard, J. P. 2002. Cloned rabbits produced by nuclear transfer from adult somatic cells. Nature Biotech. 20:366-369 https://doi.org/10.1038/nbt0402-366
  7. Cibelli, J. B., Stice, S. L., Golueke, P. J., Kane, J. J., Jerry, J., Blackwell, C., Abel Ponce de Leon, F. and Robl, J. M. 1998. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280:1256-1258 https://doi.org/10.1126/science.280.5367.1256
  8. Galat, V. V., Lagutina, I. S., Mesina, M. N., Chernech, V. J. and Prokofiev, M. I. 1999. Developmental potential of rabbit nuclear transfer embryos derived from donor fetal fibroblast. Theriogenology 51:203 (abstr.)
  9. Hill, J. R., Winger, Q. A., Long, C. R., Looney, C. R., Thompson, J. A. and 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
  10. Kato, Y., Tani, T., Sotomaru, Y., Kurokawa, K., Kato, J., Doguchi, H., Yasue, H. and Tsunoda, Y. 1998. Eight calves cloned from somatic cells of a single adult. Science 282:2095-2098
  11. Kato, Y., Tani, T. and Tsumoda, Y. 2000. Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows. J. Reprod. Fertil. 120 :231-237 https://doi.org/10.1530/reprod/120.2.231
  12. Kubota, C., Yamakuchi, H., Todoroki, J., Mizoshita, K., Tabara, N., Barber, M. and Yang, X. 2000. Six cloned calves produced from adult fibroblast cells after long-term culture. Proc. Natl. Acad. Sci. USA 97:990-995 https://doi.org/10.1073/pnas.97.3.990
  13. 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 $\alpha$-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295:1089-1092 https://doi.org/10.1126/science.1068228
  14. Onishi, A., Iwamoto, M., Akita, T., Mikawa, S., Takeda, K., Awata, T., Hanada, H. and Perry, A. C. F. 2000. Pig cloning by microinjection of fetal fibroblast nuclei. Science 289:1188-1190
  15. Polejaeva, I. A., Chen, S. H., Vaught, T. D., Page, R. L., Mullins, J., Ball, S., Dai, Y., Boone, J., Walker, S., Ayares, D. L., Colman, A. and Campbell, K. H. S. 2000. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407:86-90
  16. Schnieke, A. E., Kind, A. J., Ritchie, W. A., Mycock, K., Scoot, A. R., Ritchie, M., Wilmut, 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
  17. Wakayama, T. and Yanagimachi, R. 2001. Mouse cloning with nucleus donor cells of different age and type. Mol. Reprod. Dev. 58:376-383 https://doi.org/10.1002/1098-2795(20010401)58:4<376::AID-MRD4>3.0.CO;2-L
  18. Westhusin, M. E., Levanduski, M. J., Scarborough, R., Looney, C. R. and Bonodioli, K. R. 1992. Viable embryos and normal calves after nuclear transfer into Hoechst stained enucleated demi-oocytes of cow. J. Reprod. Fertil. 95:475-480 https://doi.org/10.1530/jrf.0.0950475
  19. Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J. and Campbell, K. H. S. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385:810-813
  20. Wells, D. N., Misica, P. M. and Tervit, H. R. 1999. Production of cloned calves following nuclear transfer with cultured adult mural granulosa cell. BioI. Reprod. 60:996-1005 https://doi.org/10.1095/biolreprod60.4.996