Korean Journal of Animal Reproduction (한국가축번식학회지)

The Korean Society of Animal Reproduction (한국동물번식학회)
권호 표지

Production of Transgenic Porcine haboring the Human Erythropoietin(EPO) Gene

사람 조혈인자 유전자(Human Erythropoietin Gene)를 도입한 형질전환돼지 생산

  • 이연근 (축산기술연구소 응용생명공학과) ;
  • 박진기 (축산기술연구소 응용생명공학과) ;
  • 민관식 (축산기술연구소 응용생명공학과) ;
  • 이창현 (축산기술연구소 응용생명공학과) ;
  • 성환후 (축산기술연구소 응용생명공학과) ;
  • 전익수 (축산기술연구소 응용생명공학과) ;
  • 임석기 (축산기술연구소 응용생명공학과) ;
  • 양병철 (축산기술연구소 응용생명공학과) ;
  • 임기순 (축산기술연구소 응용생명공학과)
  • Published : 2002.06.01
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Abstract

This study was performed during the four seasons for the production of transgenic pigs containing the human erythropoietin(hEPO) transgene. Purebred Landrace gilts and sows approximately 8∼15 months of age (n=42) were used fur the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Synchronization and superovulation procedures were used that cyclic gilts were synchronized with 20mg altrenogest (ALT) per day for 9days after PG600 administration followed by superovulation with 1500IU pregnant mares serum gonadotropin (PMSG) and 500IU human chorionic gonadotrophin (hCG). Preparation of recombinant gene for microinjection is mice whey acidic protein promoter (mWAP) linked to human erythropoietin (hEPO) gene. After hormone treatment, 650 embryos were collected from 23 donors and 83.1% (540/650) embryos were in 1-cell stage which can be visualized the pronuclei for DNA microinjection. A total of 543 DNA microinjected embryos fiom donors were transferred to 19 synchronized recipients, seven of them maintained pregnancy and delivered 47 piglets. One of the 47 offsprings were determined to have transgene by PCR analysis. The overall rate of transgenic production was 2.13% (tansgenic/offspring). This study provides the success and useful information regarding production of transgenic pig for bioreactor research.

본 연구는 사람의 조혈촉진 유전자(hEPO)가 도입된 형질전환 돼지를 생산하기 위해 사계절동안 수행하였다. 약 8∼15개월령의 순종의 랜드레이스 경산돈 및 미경산돈 42두는 유전자 미세주입을 위한 1세포기 단계의 수정란 채란 및 이식을 위해 사용하였으며, 발정동기화 및 과배란 방법은 PG 600 주입 후 9일간 매일 20mg의 altrenogest를 사료에 첨가하여 급여하였다. Altrenogest를 9일간 급여 후 1,500IU의 PMSG와 500IU의 hCG를 주입하므로서 과배란을 유도하였다. 미세주입을 위한 유전자는 mouse whey acidic protein(mWAP) 프로모터에 hEPO 유전자를 연결하여 준비하였으며, 호르몬 처리후 23두의 공란돈으로 부터 650개의 난자를 회수하였으며, 이 중 83.1%(540/650)는 DNA 미세주입을 위해 전핵을 관찰할 수 있는 1-세포기의 수정란이었다. 이중 유전자가 미세주입 된 543개의 난자를 19두의 수란돈에 이식하였으며 7두의 임신돈으로부터 47두의 자돈을 생산하였다. 생산된 자돈 47두로부터 꼬리조직으로부터 분리된 DNA의 PCR 검정 결과 수컷 1두가 형질전환 양성반응을 나타내어 2.13%의 형질전환율을 나타내었으며, 이러한 연구의 결과는 생체반응기(bioreactor)연구에 있어서 형질전환 돼지생산의 성공적이며 유용한 정보를 제공할 것으로 사료된다.

Keywords

References

  1. Aguirre, A., Castro-Palomino, N., De la Fuente, J. and Ovidio Castro F. O. 1998. Expression of human erythropoietin transgenes and of the endogenous WAP gene in the mammary gland of transgenic rabbits during gestation and lactation. Transgenic Res., 7(4):311-317 https://doi.org/10.1023/A:1008882332133
  2. Barash, I., Faermanm, A., Richenstein, M., Kari, R, Damary, G. M., Shani, M. and Bissell, M. J. 1999. In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with beta-lactoglobulin and whey acidic protein promoters Mol. Reprod. Dev., 52(3):241-252 https://doi.org/10.1002/(SICI)1098-2795(199903)52:3<241::AID-MRD1>3.0.CO;2-X
  3. Bames, F. L. 2000. The effects of the early uterine environment on the subsequent development of embryo and fetus. Theriogenology, 53: 649-658 https://doi.org/10.1016/S0093-691X(99)00264-2
  4. Bowen, R. M. R., Schnieke, A., Seidel, G., Brink, Z., Stacey, A., Thomas, W. and Kajikawa, K. 1994. Transgenic cattle resulting from biopsied embryos: Expression of c-ski in a transgenic calf. BioI. Reprod., 6:647-652
  5. Brem, G., Brenig, B., Goodman, H. M., Selden, R. C., Graf, F., Kruff, B., Springman, K., Hondele, J., MEyer, J., Winnaker, E-I. and Krausslich, H. 1985. Production of transgenic mice, rabbit and pigs by microinjection into pronuclei. Zuchthygiene, 20:251-252 https://doi.org/10.1111/j.1439-0531.1985.tb00423.x
  6. David, R. B., Blom, A. K., Sjaastad, O. V. and Harbitz, I. 2001. The porcine erythropoietin gene: eDNA sequence, genomic sequence and expression analyses in piglets. Domest. Anim. Endocrinol., 20(2):137-147 https://doi.org/10.1016/S0739-7240(01)00089-3
  7. Divoky, V., Liu, Z., Ryan, T. M., Prchal, J. F., Townes, T. M. and Prchal, J. T. 2001. Mouse model of congenital polycythemia: Homologous replacement of murine gene by mutant human erythropoietin receptor gene. Proc. Natl. Acad. Sci. USA. 98(3):986-991 https://doi.org/10.1073/pnas.98.3.986
  8. Eyestone, W. H. 1994. Challenges and progress in the production of transgenic cattle. Reprod. Fertil. Dev., 6:647-652 https://doi.org/10.1071/RD9940647
  9. Fu, P., Evans, B., Lim, G. B., Moritz, K. and Wintour, E. M. 1993. The sheep erythropoietin gene: molecular cloning and effect of hemorrhage on plasma erythropoietin and renal/liver messenger RNA in adult sheep. Mol. Cell. Endocrinol., 93(2):107-116 https://doi.org/10.1016/0303-7207(93)90113-X
  10. Funahashi, H., Stumpf, T. T., Terlouw, S. L., Cantley, T. C., Rieke, A. and Day, B. N. 1994. Developmental ability of porcine oocytes matured and fertilized in vitro. Theriogenology, 39:965-973 https://doi.org/10.1016/0093-691X(93)90433-6
  11. Gordon, J., Scagnos, G., Plotkin, D., Barbosa, J. and Ruddle, F. 1980. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. USA 77:73807384 https://doi.org/10.1073/pnas.77.12.7380
  12. Hajdu, M. A., Knight, J. W., Canseco, R. S., Krisher, R. L., Velander, W. H., Pearso, K. E. and Gwazdauskas, F. C. 1994. Effect of culture conditions, Donor age, and Injection Site on in vitro development of DNA microinjected porcine zygotes. J. Anim. Sci., 72:1299-1305 https://doi.org/10.2527/1994.7251299x
  13. 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 microinjection. Nature, 315:680-683 https://doi.org/10.1038/315680a0
  14. Hill, K., Curry, J., DeMayo, F., Jones-Diller, K., Slapak, J. and Bondioli, K. 1992. Production of transgenic cattle by pronuclear injection. Theriogenology, 37:222abstr
  15. Inuzuka, H., Yamanouchi. K., Tachi, C. and Tojo, H. 2001. A transgenic mouse model for investigating the response of the upstream region of whey acidic protein (WAP) gene to various steroid hormones. Exp. Anim., 50(1):1-7
  16. Kirby, S. L. 1999. The therapeutic potential of erythropoietin receptor transgenes. Cytokines. Cell. Mol. Ther., 5(2):97-104
  17. Kochling, J., Curtin, P. T. and Madan, A. 1998. Regulation of human erythropoietin gene induction by upstream flanking sequences in transgenic mice. Br. J. Haematol., 103(4):960-968 https://doi.org/10.1046/j.1365-2141.1998.01081.x
  18. Korhonen, V. P., Tolvanen, M., Hyttinen, J. M., Uusi-Oukari, M., Sinervirta, R., Alhonen, L., Jauhiainen, M., Janne, O. A. and Janne, J. 1997. Expression of bovine beta-lactoglobulin/human erythropoietin fusion protein in the milk of transgenic mice and rabbits. Eur. J. Biochern., 245(2):482-489
  19. Kubisch, H. M., Larson, M. A., Funahashi, H., Day, B. N. and Roberts, R. M. 1995. Pronuclear visibility, development and transgene expression in IVMlIVF-derived porcine embryos. Theriogenology, 44:391-401 https://doi.org/10.1016/0093-691X(95)00193-C
  20. Lathe, R., Clark, A., Archibald, A., Bishop, JI, Simons, P. and Wilmut, 1985. I.Novel products from livestock. In: smith, C., King, W. McKay, J.,(eds). Exploiting new technology in animal breeding . Oxford: Science Publications 99-102
  21. Lee, T. H., Kim, S. J., Han, Y. M., Yu, D. Y., Lee, C. S., Choi, Y. J, Moon, H. B., Baik, M. G. and Lee, K. K. 1998. Matrix attachment region sequences enhanced the expression frequency of a whey acidic protein/human lactoferrin fusion gene in the mammary gland of transgenic mice. Mol. Cells., 8(5):530-536
  22. Lin, F. K., Lin, C. H., Lai, P. H., Browne, J K., Egrie, J. C., Smalling, R., Fox; G. M., Chen, K., Castro, M. and Suggs, S. 1986. Monkey erythropoietin gene: Cloning, expression and comparison with the human erythropoietin gene. Gene, 44:201-209 https://doi.org/10.1016/0378-1119(86)90183-6
  23. Lin, F. K., Suggs, S., Lin, C. H., Browne, J K., Smalling, R., Egrie, J. C., Chen, K., Fox, G. M., Martin, F., Stabinsky, Z., Badrawi, S. M., Lai, P. H. and Goldwasser, E. 1985. Cloning and expression of the human erythropoietin gene. Proc. Natl. Acad. Sci. U.S.A. 82 (22): 7580-7584
  24. Massoud, M., Attal, J, Thepot, D., Pointu, H., Stinnakre, M. G., Theron, M. C., Lopez, C. and Houdebine, L. M. 1996. The deleterious effects of human erythropoietin gene driven by the rabbit whey acidic protein gene promoter in transgenic rabbits. Reprod. Nutr. Dev., 36(5): 555-563 https://doi.org/10.1051/rnd:19960511
  25. Mikus, T., Maly, P., Poplstein, M., Landa, V., Trefil, P. and Lidicky, J. 2001. Expression of human erythropoietin gene in the mammary gland of a transgenic mouse. Folia Bioi (praha), 47(6):187-195
  26. Nagao, M., Suga, H., Okano, M., Masuda, S., Narita, H., Ikura, K. and Sasaki, R. 1992. Nucleotide sequence of rat erythropoietin. Biochim. Biophys. Acta, 1171(1):99-102
  27. Palmiter, R, Brinster, R, Hammer, R., Trubaner, M., Rosenfeld, M., Bimberg, N. and Evans, R. 1982. Dramatic growth of mice that developed from eggs microinjected with metallothionine growth hormone fusion genes. Nature, 300:611-615 https://doi.org/10.1038/300611a0
  28. Pursel, V. G. and Wall, R. J. 1996. Effect of transferred ova per recipient and dual use of donors as recipients on production of transgenic swine. Theriogenology, 46:201-209 https://doi.org/10.1016/0093-691X(96)00178-1
  29. Rodriguez, A., Castro, F. O., Aguilar, A., Ramos, B., Del Barco, D. G., Lleonart, Rand De la Fuente, J. 1995. Expression of active human erythropoietin in the mammary gland of lactating transgenic mice and rabbits. Biol. Res., 28(2):141-153
  30. Sambrook, J. and Russell, D. W. 1991. Molecular Cloning. A laboratory manual.(3rd Ed). Cold Spring Harbor Laboratory Press
  31. Schlieper, B. and Holtz, W. 1986. Transfer of pig embryos collected by laparotomy or slaughter. Anim. Reprod. Sci., 12:109-114 https://doi.org/10.1016/0378-4320(86)90050-3
  32. Semenza, G. L., Dureza, R. C., Traystman, M. D., Gearhart, J. D. and Antonarakis, S. E. 1990. Human erythropoietin gene expression in transgenic mice: multiple transcription initiation sites and cis-acting regulatory elements. Mol. Cell. Biol., 10(3):930-938 https://doi.org/10.1128/MCB.10.3.930
  33. Semenza, G. L., Traystman, M. D., Gearhart, J. D. and Antonarakis, S. E. 1989. Polycythemia in transgenic mice expressing the human erythropoietin gene. Proc. Natl. Acad. Sci. USA 86(7):2301-2305 https://doi.org/10.1073/pnas.86.7.2301
  34. Shoemaker, C. B. and Mitsock, L. D. 1986. Murine erythropoietin gene: Cloning, expression, and human gene homology. Mol. Cell. Biol., 6:849-858 https://doi.org/10.1128/MCB.6.3.849
  35. Soede, N. M. Noordhuizen, P. P. T. M. and Kemp, B. 1992. The duration of ovulation in pigs, studied by transrectal ultrasonography, is not related to early embryonic diversity. Theriogenology, 38:653
  36. Van Cott, K. E., Lubon, H., Russell, C. G., Butler, S. P., Gwazdauskas, F. C., Knight, J., Drohan, W. N. and Velander, W. H. 1997. Phenotypic and genotypic stability of multiple lines of transgenic pigs expressing recombinant human protein C. Transgenic Res., 6(3):203-212 https://doi.org/10.1023/A:1018442124584
  37. Wall, R. 1996. Modification of milk composition in transgenic animals. American Society of Animal Science. 165-188
  38. Wall, R. J., Pursel, V. G., Shamay, R. A., McKinght., Pittius, C. W. and Hennighausen, L. 1991. High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine. Proc. Nati. Acad. Sci. USA 88: 1696
  39. Wall, R. J., Pursel, V. G., Hammer, R. E. and Brinster, R. L. 1985. Development of porcine ova that were centrifuged to permit visualization of pronuclei and nuclei. Biol. Reprod., 32:645-651 https://doi.org/10.1095/biolreprod32.3.645
  40. Wall, R. J., Rexroad, C. E. Jr., Powell, A., Shamay, A., McKnight, R. and Hennighausen, L. 1996. Synthesis and secretion of the mouse whey acidic protein in transgenic sheep. Transgenic Res., 5(1):67-72
  41. Wei, Q., Fan,J1. and Chen, D. 1993. Effect of number of transferred microinjected embryos on pregnancy rate and litter size of pigs. Theriogenology, 39:338 abstr
  42. White, K. L., Southern, L. L., Rickords, L. F. and Wood, T. C. 1988. Embryonic development and quality in cycling crossbreed gilts following altrenogest synchronization and exogenous gonadotrophin administration. Theriogenology, 29:326 abstr
  43. Williams, B. L., Sparks, A. E. T., Canseco, R. S., Knight, J. W., Johnson, J. L., Velander, W. H., Page, R. L., Drohan, W. N., Kornegay, E. T., Pearson, R. E., Wilkins, T. D. and Gwazdauskas, F. C. 1992a. Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer. Theriogenology, 38:501511
  44. Williams, B. L., Sparks, A. E. T., Canseco, R. S., Knight, J. W., Johnson, J. L., Velander, W. H., Page, R. L., Drohan, W. N., Young, J. M., Pearson, R. E., Wilkins, T. D. and Gwazdauskas, F. C. 1992b. In vitro development of zygotes from prepubertal gilts after microinjection of DNA. J. Anim. Sci., 70:2207-221