Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지

Efficient Culture of Porcine Fetal Fibroblasts

돼지 태아 섬유아 세포의 효과적인 배양

  • Kim, H.M. (Department of Animal Science, Insti. of Ag. Sci. and Tech., College of Agriculture & Life Science, Chonnam National University) ;
  • Lee, S.M. (Department of Animal Science, Insti. of Ag. Sci. and Tech., College of Agriculture & Life Science, Chonnam National University) ;
  • Park, H.Y. (Department of Animal Science, Insti. of Ag. Sci. and Tech., College of Agriculture & Life Science, Chonnam National University) ;
  • Moon, S.J. (Department of Animal Science, Insti. of Ag. Sci. and Tech., College of Agriculture & Life Science, Chonnam National University) ;
  • Kang, M.J. (Department of Animal Science, Insti. of Ag. Sci. and Tech., College of Agriculture & Life Science, Chonnam National University)
  • 김혜민 (전남대학교 농업생명과학대학, 농업과학기술연구소, 동물자원학부) ;
  • 이상미 (전남대학교 농업생명과학대학, 농업과학기술연구소, 동물자원학부) ;
  • 박효영 (전남대학교 농업생명과학대학, 농업과학기술연구소, 동물자원학부) ;
  • 문승주 (전남대학교 농업생명과학대학, 농업과학기술연구소, 동물자원학부) ;
  • 강만종 (전남대학교 농업생명과학대학, 농업과학기술연구소, 동물자원학부)
  • Published : 2007.09.29
Download PDF
⟨ Previous Next ⟩

Abstract

Culture method of somatic cells is one of the important factors in the production of transgenic pigs by somatic cell nuclear transfer. In this study, we established an efficient culture method of porcine fetal fibroblasts. Porcine fetal fibroblasts were isolated from 33-day-old fetuses. The proliferation of porcine fetal fibroblasts was analyzed by different serum types and culture media. The cultures in medium supplied 15% ES screened FBS showed faster increase in cell number than 15% FBS. Also, fetal fibroblasts have been propagated continuously for $7{\sim}8$ passages in ES modified DMEM and DMEM medium. We transfected $PGK-neo^r$ vector (pKJ2) into porcine fetal fibroblasts to estimate colony formation in this culture condition. The formation of colonies was confirmed in the medium containing $300\;{\mu}g/ml$ G418 at 12 day. These data show that this culture system can be used screening of porcine somatic cells transfected transgene.

체세포의 배양 방법은 체세포 핵이식에 의한 형질 전환 돼지 생산에 있어서 중요한 요인 중 하나이다. 본 연구에서는 돼지 태아 섬유 아세포의 효율적인 배양 방법을 수려하였다. 돼지 태아 섬유 아세포는 임신 33일째 태아로부터 제조하였으며, 돼지 태아 섬유아세포의 증식을 혈청과 배지 종류별로 분석하였다. 그 결과, 15% ES screened FBS가 포함된 DMEM 배지에서의 배양은 15% FBS보다 세포수의 증가가 훨씬 더 빠르게 나타났다. 또한, 태아 섬유아 세포는 DMEM/F-12와 다르게 ES midified DMEM과 DMEM 배지에서 $7{\sim}8$번째 계대까지 증식이 유지되었다. 이러한 배양 조건에서 PGK-neo 벡터(pKJ2)를 돼지 태아 섬유아 세포에 도입한 다음 12일간 $300\;{\mu}g/ml$ G418이 포함된 배지에서 선별하여 colony를 얻을 수 있었다. 이러한 결과는 본 연구에 이용된 배양 시스템이 transgenic vector를 도입시킨 돼지 체세포의 screening에 이용될 수 있음을 보여주고 있다.

Keywords

References

  1. Boquest AC, Day BN and Prather RS. 1999. Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells. Biol. Reprod., 60:1013-1019 https://doi.org/10.1095/biolreprod60.4.1013
  2. Boquest AC, Grupen CG, Harrison SJ, McIlfatrick SM, Ashman RJ, d'Apice AJ and Nottle MB. 2002. Production of cloned pigs from cultured fetal fibroblast cells. Biol. Reprod., 66:1283-1287 https://doi.org/10.1095/biolreprod66.5.1283
  3. Cheong HT, Ikeda K, Martinez Diaz MA, Katagiri S and Takahashi Y. 2000. Development of reconstituted pig embryos by nuclear transfer of cultured cumulus cells. Reprod. Fertil. Dev., 12:15-20 https://doi.org/10.1071/RD00051
  4. Cheong HT, Park KW, Im GS, Lai L, Sun QY, Day BN and Prather RS. 2002. Effect of elevated $Ca^{(2+)}$ concentration in fusion/activation medium on the fusionand development of porcine fetal fibroblast nuclear transfer embryos. Mol. Reprod. Dev., 61:488-492 https://doi.org/10.1002/mrd.10110
  5. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA and Robl JM. 1998. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science, 280:1256-1258 https://doi.org/10.1126/science.280.5367.1256
  6. Cui W, Wylie D, Aslam S, Dinnyes A, King T, Wilmut I and Clark AJ. 2003. Telomerase-immortalized sheep fibroblasts can be reprogrammed by nuclear transferto undergo early development. Biol. Reprod., 69: 15-21 https://doi.org/10.1095/biolreprod.102.013250
  7. Freshney RI. 2000. Culture of animal cells; a manual of basic technique. 4th ed., Willy-Liss, New York, pp. 99-103
  8. Kato Y, Tani T and Tsunoda 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
  9. 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
  10. Kuhholzer B, Tao T, Machaty Z, Hawley RJ, Greenstein JL, Day BN and Prather RS. 2000. Production of transgenic porcine blastocysts by nuclear transfer. Mol. Reprod. Dev., 56: 145-148 https://doi.org/10.1002/(SICI)1098-2795(200006)56:2<145::AID-MRD4>3.0.CO;2-8
  11. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ and Prather RS. 2002a. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfercloning. Science, 2002a 295:1089-1092 https://doi.org/10.1126/science.1068228
  12. Lai L, Park KW, Cheong HT, Kuhholzer B, Samuel M, Bonk A, Im GS, Rieke A, Day BN, Murphy CN, Carter DB and Prather RS. 2002b. Transgenic pig expressing the enhanced green fluorescent protein produced bynuclear transfer using colchicine-treated fibroblasts as donor cells. Mol. Reprod. Dev., 62:300-306 https://doi.org/10.1002/mrd.10146
  13. Mir B and Piedrahita JA. 2004. Nuclear localization signal and cell synchrony enhance gene targeting efficiency in primary fetal fibroblasts. Nucleic Acids Res., 32:e25 https://doi.org/10.1093/nar/gnh023
  14. Park KW, Cheong HT, Lai L, Im GS, Kuhholzer B, Bonk A, Samuel M, Rieke A, Day BN, Murphy CN, Carter DB and Prather RS. 2001. Production of nuclear transfer-derived swine that express the enhanced greenfluorescent protein. Anim. Biotechnol., 12:173-181 https://doi.org/10.1081/ABIO-100108344
  15. Park KW, Lai L, Cheong HT, Cabot R, Sun QY, Wu G, Rucker EB, Durtschi D, Bonk A, Samuel M, Rieke A, Day BN, Murphy CN, Carter DB and Prather RS. 2002. Mosaic gene expression in nuclear transfer-derived embryos and the production of cloned transgenic pigs from ear-derived fibroblasts. Biol. Reprod., 66:1001-1005 https://doi.org/10.1095/biolreprod66.4.1001
  16. Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie M, Wilmut I, Colman A and Campbell KH. 1997. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science, 278:2130-2133 https://doi.org/10.1126/science.278.5346.2130
  17. Wilmut I, Schnieke AE, McWhir J, Kind AJ and Campbell KH. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature, 385:810-813 https://doi.org/10.1038/385810a0
  18. Zakhartchenko V, Alberio R, Stojkovic M, Prelle K, Schernthaner W, Stojkovic P, Wenigerkind H, Wanke R, Duchler M, Steinborn R, Mueller M, Brem G and Wolf E. 1999. Adult cloning in cattle: potential of nuclei from a permanent cell line and from primary cultures. Mol. Reprod. Dev., 54:264-272 https://doi.org/10.1002/(SICI)1098-2795(199911)54:3<264::AID-MRD7>3.0.CO;2-Y