Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지

Development of Transgenic NT Embryos Using Bovine Fetal Fibroblasts Transfected with hFSH Gene

hFSH 유전자가 도입된 소 태아섬유아세포를 이용한 형질 전환 복제 수정란의 발달

  • Yang B.C. (National Livestock Research Institute, RDA) ;
  • Im G.S. (National Livestock Research Institute, RDA) ;
  • Kim D.H. (National Livestock Research Institute, RDA) ;
  • Min K.S. (Animal Biotechnology, Graduate School of Bio. and Information Technology, Hankyong National University) ;
  • Yoon D.H. (National Livestock Research Institute, RDA) ;
  • Park H.S. (National Livestock Research Institute, RDA) ;
  • Kim S.W. (National Livestock Research Institute, RDA) ;
  • Hwang I.S. (National Livestock Research Institute, RDA) ;
  • Seo J.S. (National Livestock Research Institute, RDA) ;
  • Seong H.H. (National Livestock Research Institute, RDA) ;
  • Yang B.S. (National Livestock Research Institute, RDA)
  • Published : 2006.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

The purpose of this study was to develope the transgenic cattle expressing hFSH into the urine using the nuclear transfer. To produce the interest gene in urine, the specific vector was ligated with hFSH gene undo. maUII promoter. The fetal fibroblast cells (KbFF) were isolated from a 45-day male fetus. The hFSH gene was co-transfected with pcDNA3 (neo) vector to KbFF cells by electroporation. The gene-transfected cells were cultured with G-418 selection medium for 2 weeks. Selected colonies were confirmed by PCR. For nuclear transfer, enucleated bovine oocytes were transferred with hFSH transfected or nontransfected fetal fibroblasts. The cleavage and blastocyst formation rates were significantly lower (p<0.05) in cloned embryos transfected with hFSH gene (68.7% and 15.7%) than in those non-transfected (67.6% and 24.5 %), respectively. Apoptosis analysis showed no difference between hFSH transfected and non-transfected blastocysts (p>0.05). The blastocysts were transfected to 77 (control 24, hFSH 53) recipient cows. Two calves were born (1.9%) following transfer with NT embryos transfected with hFSH gene, but they were confirmed not to be transgenic calves. This result shows that the hFSH colonies were mixed with transfected and non transfected cells. Further research will be needed for selection and establishment of gene transfected cells.

본 연구의 목적은 요를 통해 hFSH를 발현하는 형질 전환 소의 생산이다. 요의 분비와 관련 있는 유전자로서 mUII promoter를 사용하여 hFSH유전자를 구성했다. 태아섬유아세포(KbFF)는 임신 45일령의 태아(male)에서 채취하였다. hFSH gene은 pcDNA3(neo) vector와 같이 KbFF 세포에 electroporation 방법으로 transfection하였다. 유전자를 transfection한 세포는 G-418로 2주 동안 배양하였고, 선발된 colony는 PCR로 확인하였다. 핵이 제거된 난자는 hFSH가 transfection된 세포와 transfection 되지 않은(control) 세포를 이용하여 핵이식하였다. 48시 간 후 hFSH가 transfection된 세포는 68.7%의 수정란이 난할되었으며, 8일 후 15.7%의 수정란이 배반포로 발달하였다. 그러나 대조구에서는 67.6%가 난할되었으며, 24.5%가 배반포로 각각 발달하였다. 이들 배반포에서 apoptosis 분석 결과 hFSH 유전자가 transfection된 또는 transfection되지 않은 대조구에서 유의적인 차이는 보이지 않았다. 배반포는 53두의 수란우에 이식하여 두 마리의 산자가 생산되었으나(1.9%) hFSH가 transfection되지 않은 것으로 나타났다. 이 결과는 선발된 hFSH colony에서 transfection되지 않은 세포가 혼합되어 있었다는 것을 나타내 주고 있으며, colony의 선발과 검증에 더 많은 연구의 필요성이 있음을 나타내준다.

Keywords

References

  1. Bordignon V, Keyston R, Lazaris A, Bilodeau AS, Pontes JHF, Arnold D, Fecteau G, Keefer C and Smith LC. 2003. Transgene expression of green fluorescent protein and germ line transmission in cloned calves derived from in vitro-transfected somatic cells. Bio. Reprod., 68:2013-2023 https://doi.org/10.1095/biolreprod.102.010066
  2. Cappello F, Stassi G, Lazzereschi D, Renzi L, Di Stefano C, Marfe G, Giancotti P, Wang HJ, Stoppacciaro A, Forni M, Bacci ML, Turchi V, Sinibaldi P, Rossi M, Bruzzone P, Pretagostini R, Della Casa G, Cortesini R, Frati Land Lavitrano M. 2000. hDAF expression in hearts of transgenic pigs obtained by sperm-mediated gene transfer. Transplant. Proc., Aug 32(5): 895-896
  3. Chan A W, Chong KY, Martinovich C, Simerly C and Schatten G. 2001. Transgenic monkeys produced by retroviral gene transfer into mature oocytes. Science, 291(5502):309-312 https://doi.org/10.1126/science.291.5502.309
  4. Chen SH, Vaught TD, Monahan JA, Boone J, Emslie E, Jobst PM, Lamborn AE, Schnieke A, Robertson L, Colman A, Dai Y, Polejaeva IA and Ayares DL. 2002. Efficient production of transgenic cloned calves using preimplantation screening. Biol. Reprod., Nov 67(5):1488-1492 https://doi.org/10.1095/biolreprod.102.006981
  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, May 22. 280(5367): 1256-1258 https://doi.org/10.1126/science.280.5367.1256
  6. Enright BP, Taneja M, Schreiber D, Riesen J, Tian XC, Fortune JE and Yang X. 2002. Reproductive characteristics of cloned heifers derived from adult somatic cells. Biol. Reprod., Feb 66(2):291-296 https://doi.org/10.1095/biolreprod66.2.291
  7. Fan W, Plaut K, Bramley AJ, Barlow JW and Kerr DE. 2002. Adenoviral-mediated transfer of a lysostaphin gene into the goat mammary gland. J. Dairy Sci., Jul 85(7):1709-1716 https://doi.org/10.3168/jds.S0022-0302(02)74244-6
  8. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA and Ruddle FH. 1980. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. USA., Dec 77(12):7380-7384
  9. Hill JR, Roussel AJ, Cibelli JB, Edwards JF, Hooper NL, Miller MW, Thompson JA, Looney CR, Westhusin ME, Robl JM and Stice SL. 1999. Clinical and pathologic features of cloned transgenic calves and fetuses (13 case studies). Theriogenology, Jun 51(8):1451-1465
  10. Keefer CL, Baldassarre H, Keyston R, Wang B, Bhatia B, Bilodeau AS, Zhou JF, Leduc M, Downey BR, Lazaris A and Karatzas CN. 2001. Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes. Biol. Reprod., Mar 64(3):849-856 https://doi.org/10.1095/biolreprod64.3.849
  11. Lee GS, Kim HS, Hyun SH, Lee SH, Jeon HY, Nam DH, Jeong YW, Kim S, Kim JH, Han JY, Ahn C, Kang SK, Lee BC and Hwang WS. 2005. Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein. Theriogenology, Mar 1 63(4):973-991 https://doi.org/10.1016/j.theriogenology.2004.04.017
  12. Noble MS, Rodriguez-Zas S, Cook JB, Bleck GT, Hurley WL and Wheeler MB. 2002. Lactational performance of first-parity transgenic gilts expressing bovine alpha-lactalbumin in their milk. J. Anim. Sci., Apr 80(4):1090-1096
  13. Perry AC, Wakayama T, Kishikawa H, Kasai T, Okabe M, Toyoda Y and Yanagimachi R. 1999. Mammalian transgenesis by intracytoplasmic sperm injection. Science, May 14 284(5417):1180-1183 https://doi.org/10.1126/science.284.5417.1180
  14. Sin FY, Walker SP, Symonds JE, Mukherjee UK, Khoo JG and Sin IL. 2000. Electroporation of salmon sperm for gene transfer: Efficiency, reliability, and fate of transgene. Mol. Reprod. Dev., Jun 56(S2):285-288 https://doi.org/10.1002/(SICI)1098-2795(200006)56:2+<285::AID-MRD16>3.0.CO;2-4
  15. Zakahartchenko V, Mueller S, Alberio R, Schernthaner W, Stojkovic M, Wenigerkind H, Wanke RD, Lassnig 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