Production of Bovine Transgenic Cloned Embryos using Prourokinase-Transfected Somatic Cells: Effect of Expression Level of Reporter Gene

인간 Prourokinase가 도입된 체세포를 이용한 소 형질전환 복제란 생산: 표지유전자 발현정도에 따른 효과

  • J. K. Cho (Department of Theriogenology & Biotechnology, College of Veterinary Medicine, Seoul National University) ;
  • M.M.U. Bhuiyan (Department of Theriogenology & Biotechnology, College of Veterinary Medicine, Seoul National University) ;
  • G. Jang (Department of Theriogenology & Biotechnology, College of Veterinary Medicine, Seoul National University) ;
  • G. Jang (Department of Theriogenology & Biotechnology, College of Veterinary Medicine, Seoul National University) ;
  • Park, E. S. (School of Agriculutral Biotechnology, Seoul National University) ;
  • S. K. Kang (Department of Theriogenology & Biotechnology, College of Veterinary Medicine, Seoul National University) ;
  • Lee, B. C. (Department of Theriogenology & Biotechnology, College of Veterinary Medicine, Seoul National University) ;
  • W. S. Hwang (Department of Theriogenology & Biotechnology, College of Veterinary Medicine, Seoul National University)
  • Published : 2002.08.01

Abstract

Human Prourokinase (proUK) offers potential as a novel agent with improved fibrin specificity and, as such, may offer advantages as an attractive alternative to urokinase that is associated with clinical benefits in patients with acute peripheral arterial occlusion. For production of transgenic cow as human proUK bioreacotor, we conducted this study to establish efficient production system for bovine transgenic embryos by somatic cell nuclear transfer (NT) using human prourokinase gene transfected donor cell. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human prourokinase target gene into a pcDNA3 plasmid. Cumulus cells were used as donor cell and transfected with the expression plasmid using the Fugene 6 as a carrier. To increase the efficiency for the production of transgenic NT, development rates were compared between non-transfected and transfected cell in experiment 1, and in experiment 2, development rates were compared according to level of GFP expression in donor cells. In experiment 1, development rates of non-transgenic NT embryos were significantly higher than transgenic NT embryos (43.3 vs. 28.4%). In experiment 2, there were no significant differences in fusion rates (85.4 vs. 78.9%) and cleavage rates (78.7 vs. 84.4%) between low and high expressed cells. However, development rates to blastocyst were higher in low expressed cells (17.0 vs. 33.3%), and GFP expression rates in blastocyst were higher in high expressed cells (75.0 vs. 43.3%), significantly.

Keywords

References

  1. Aguirre A, Castro-Palomino N, Fuente JDA and Castro FO. 1998. Expression of human erythropoietin transgenes and of the endogenous wap gene in the mammary gland of transgenic rabbits during gestation and lactation. Transgenie Res., 7:311-317 https://doi.org/10.1023/A:1008882332133
  2. Arat S, Rzucidlo SJ, Gibbons J, Miyoshi K and Stice SL. 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
  3. Archibald AL, McClenaghan M, Valerie H, Simons JP and Clark AJ. 1990. High-level expression of biologically active human ($\alpha$ l-antitrypsin in the milk of transgenic mice. Proc. Natl. Acad. Sci., 87: 5178-5182 https://doi.org/10.1073/pnas.87.13.5178
  4. Chan AWS. 1999. Transgenic animals: Current and alternative strategies. Cloning, 1:25-46 https://doi.org/10.1089/15204559950020076
  5. Cho JK, Lee BC, Park JI, Lim JM, Shin SJ, Kim KY, Lee BD and Hwang WS. 2002. Development of bovine oocytes reconstructed with different donor somatic cells with or without serum starvation. Theriogenology, 15:1819-1828
  6. 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 non-quiescent fetal fibroblasts. Science, 280: 1256-1258 https://doi.org/10.1126/science.280.5367.1256
  7. Hadjantonakis AK, Gertsenstein M, Ikawa M, Okabe M and Nagy A. 1998. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech. Dev., 76:79-90 https://doi.org/10.1016/S0925-4773(98)00093-8
  8. Hanazono Y, Yu JM, Dunbar CE and Emmons RVB. 1997. Green fluorescent protein retroviral vectors: low titer and high recombination frequency suggest a selective disadvantage. Hum. Gene. Therapy, 8:1313-1319 https://doi.org/10.1089/hum.1997.8.11-1313
  9. Hill JR, Burghardt RC, Jones K, Long CR, Looney CR, Shin T, Spencer TE, Thompson JA, Winger QA and Westhusin ME. 2000. Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biol. Reprod., 63:1787-1794 https://doi.org/10.1095/biolreprod63.6.1787
  10. Houdebine LM. 2000. Transgenic animal bioreactors. Transgenic Res., 9:305-320 https://doi.org/10.1023/A:1008934912555
  11. Kono T. Nuclear transfer and reprogramming. Rev of Reprod., 1997. 2:74-80 https://doi.org/10.1530/ror.0.0020074
  12. Lee BC, Yoon KY, Kim HI, Roh SH and Hwang WS. 1994. Transvaginal ultrasound-guided ovum pick-up in cattle: Effectes of estrus. Korean J. Anim. Sci., 72:434-437 https://doi.org/10.2527/1994.722434x
  13. Petters RM and Sommer JR. 2000. Transgenic animals as models for human disease. Transgenic Res., 9:347-351 https://doi.org/10.1023/A:1008926303533
  14. Platenburg GJ, Kooteijk EP, Kooiman PM, Woloshuk SL, Nuijens JH and Krimpenfort PJ, Pieper FR, de Boer HA, Strijker R. 1994. Expression of human lactoferrin in milk of transgenic mice. Transgenic Res., 3:99-108 https://doi.org/10.1007/BF01974087
  15. Rosenkrans CFJ and First NL. 1991. Culture of bovine zygotes to the blastocyst stage: Effect of amino acids and vitamins. Theriogenology, 35:266 https://doi.org/10.1016/0093-691X(91)90242-6
  16. Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie M, Wilmut I, Colman A and Campbell KHS. 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. Shani M, Barash I, Nathan M, Ricca G, Searfoss GH, Dekel I, Faerman A, Givol D and Hurwitz DR. 1992. Expression of human serum albumin in the milk of transgenic mice. Transgenic Res., 1:195-208 https://doi.org/10.1007/BF02524750
  18. Takahashi Y and First NL. 1992. In vitro development of bovine one-cell embryos: Influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology, 37:963-978 https://doi.org/10.1016/0093-691X(92)90096-A
  19. Tanahashi N and Fukuuchi Y. 2002. Treatment of acute ischemic stroke: recent progress. Intern. Med., 41:337-344 https://doi.org/10.2169/internalmedicine.41.337
  20. Van Cott KE, Lubon H, Gwazdauskas FC, Knight J, Drohan WN and Velander WH. 2001. Recombinant human protein C expression in the milk of transgenic pigs and the effect on endogenous milk immunoglobulin and transferrin levels. Transgenic Res., 10:43-51 https://doi.org/10.1023/A:1008963817646
  21. Wells DN, Misica PM and Tervit HR. 1999. Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. Biol. Reprod., 60:996-1005 https://doi.org/10.1095/biolreprod60.4.996
  22. Wilmut I, Schnieke AE, McWhir J, Kind AJ and Campbell KHS. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature, 385:810-813 https://doi.org/10.1038/385810a0
  23. Zakhartchenko V, Mueller S, Alberio R, Schernthaner W, Stojkovic M, Wenigerkind H, Wanke R, 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