Korean Journal of Animal Reproduction (한국가축번식학회지)

The Korean Society of Animal Reproduction (한국동물번식학회)
권호 표지

Transfer and Expression of the Recombinant hFSH Gene in CHO Cells and Transgenic Chickens using Retrovirus Vector System

CHO 세포와 형질전환 닭에 있어서 Retrovirus Vector System에 의한 hFSH 재조합 유전자의 전이와 발현

  • 권모선 (대구가톨릭대학교 의과대학 생리학교실) ;
  • 구본철 (대구가톨릭대학교 의과대학 생리학교실) ;
  • 심호섭 (단국대학교 의과대학 생리학교실) ;
  • 박창식 (충남대학교 동물자원학부, 충남대학교 형질전환 복제돼지 연구센터) ;
  • 이성호 (공주대학교 영상보건대학, 충남대학교 형질전환 복제돼지 연구센터) ;
  • 김태완 (충남대학교 형질전환 복제돼지 연구센터)
  • Published : 2003.09.01
Download PDF
⟨ Previous Next ⟩

Abstract

hFSH (human follicle stimulating hormone) is heterodimer consisting of $\alpha$ and $\beta$ subunits. Since assembly of the both subunits in the cell is often the rate-limiting step in production of functional hormone, single-chain hormones have been engineered by genetically linking two different cDNA fragments with a linker sequence. Using retrovirus vector system, the resulting recombinant hFSH gene was transferred in CHO cells and chicken embryos, and the expression of the gene was investigated. In CHO cells, protein synthesis from the single-chain FSH gene was 17 fold higher than that from the heterodimeric counterpart. In the study of transgenic chickens, ten of the eleven chicks hatched from 62 embryos manupulated with recombinant retrovirus stock was determined to carry transgenic genes. RT-PCR analyses confirmed transcription of the single-chain FSH gene, however, no recombinant FSH was detected from the blood samples.

hFSH는 $\alpha$와 $\beta$ subunit으로 구성된 heterodimer로서 두 subunit의 조합은 활성을 지닌 호르몬의 생산에 있어서 매우 중요한 단계이다. 이 조합과정의 효율을 증대하기 위하여 본 연구에서는 hFSH를 단일사슬의 단백질로 구축하고자 하였으며, 이의 일환으로 각 subunit 대한 cDNA단편을 연결하는 서열로 CTP linker를 도입하였다. 재조합한 hFSH-CTP 유전자는 pseudotype의 retrovirus vector system을 이용하여 CHO 세포와 닭의 배로 각각 전이되었다. CHO 세포에서의 FSH의 생산은 $\alpha$와 $\beta$를 각각 전이한 경우에 비해 hFSH-CTP를 전이한 경우에서 17배 이상 높은 것으로 나타났다. 닭에서는 유전자 전이를 시도한 62개체 중에서 11마리가 부화하였으며 그 중 10마리가 형질전환된 닭인 것으로 RT-PCR을 통하여 확인되었다. 그러나 개체의 혈중 FSH의 생산은 확인하지 못하였다. 이상의 실험 결과를 바탕으로 하여 재조합된 hFSH-CTP는 FSH의 발현에 매우 효율적인 구조로 생각되며, 또한 retrovirus를 이용한 유전자 전이 방법은 형질전환 가금의 생산에 있어서 매우 적절한 방법으로 사료된다.

Keywords

References

  1. Arora, N., Williamson, L. C., Leppla, S. H. and Halpern, J. L. 1994. Cytotoxic effects of a chimeric protein consisting of tetanus toxin light chain and anthrax toxin lethal factor in non-neuronal cells. J. Biol. Chem. 269: 26165-26171
  2. Hard, K., Mekking, A., Damm, J. B. L., Kamerling, J. P., De Boer, W., Wijnands, R. A., and Vliegenthart, J. F. G. 1990. Isolation and structure determination of the intact sialylated N-linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells. Eur. J. Biochem. 193: 263-271
  3. Keene, J. L., Matzuk, M. M., Otani, T., Fauser, B. C. J. M., Galway, A. B., Hsueh, A. J. W., and Boime, I. 1989. Expression of biologically active human follitropin in Chinese hamster ovary cells. J. BioI. Chem. 264: 4769-4775
  4. Kim, T., Lee, Y. M., Lee, H. T., Heo, Y. T., Yom, H. C., Kwon, M. S., Koo, B. C., Whang, K., and Roh, K. S. 2001. Expression of the E. coli LacZ gene in chicken embryos using replication defective retroviral vectors pakaged with vesicular stomatitis virus G glycoprotein envelopes. Asian-Aust, J. Anim. Sci. 14:163-169 https://doi.org/10.5713/ajas.2001.163
  5. Klein, J., Lobel, L., Pollak, S., Ferin, M., Xiao, E., Sauer, M. and Lustbader, J. W. 2002. Pharmacokinetics and pharmacodynamics of single-chain recombinant human follicle-stimulating hormone containing the human chorionic gonadotropin carboxyterminal peptide in the rhesus monkey. Fertil. Steril. 77: 1248-1255 https://doi.org/10.1016/S0015-0282(02)03113-8
  6. Lin, S., Galiano, N., Culp., Burns, J. C, Friedmann, T., Yee, J. K. and Hopkins, N. 1994. Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish. Science 265: 666-668
  7. Mannaerts, B., Leeuw, R. de, Ravestein, A. Van, Wezenbeek, P. Van, Schuurs, A. and Kloosterboer, H. 1991. Comparative in vitro and in vivo studies on the biological characteristics of recombinant human follicle-stimulating hormone. Endocrinology 129: 2623-2630
  8. Matzuk, M. M., Hsueh, A. J., LapoIt, P., Tsafriri, A., Keene, J. L. and Boime, I. 1990. The biological role of the carboxyl-terminal extension of human chorionic gonadotropin beta-subunit. 1990. Endocrinology 126:376-383
  9. Muyan, M. and Boime, I. 1998. The carboxyl-terminal region is a determinant for the intra-cellular behavior of the chorionic gonadotropin $\beta$ subunit: effects on the processing of the Asn-linked olighosaccharides. Mol. Endocrinol. 12: 766-772
  10. Pierce, J. and Parsons, T. 1981. Glycoprotein hormones: structure and function. Annu. Rev. Biochem. 50: 465-495
  11. Rohdewohld, H., Weiher, H., Reik, W., Jaennisch, R. and Breindi, M. 1987. Retrovirus integration and chromatin structure: Moloney murine leukemia proviral integration sites map near DNase I-hypersensitive sites. J. Virol. 61: 336-343
  12. Sano, T., Glazer, A. N. and Cantor, C. R. 1992. A streptavidin-metallothionein chimera that allows specific labeling of biological materials with many different heavy metal ions. Proc.. Natl. Acad. Sci. USA 89: 1534-1538 https://doi.org/10.1073/pnas.89.5.1534
  13. Sugahara, T., Pixley, M. R., Minami, S., Perlas, E., Ben-Menahem, D., Hsueh, A. J. W. and Boime, I. 1995. Biosynthesis of a biologically active single peptide chain containing the human common and chorionic gonadotropin subunits in tandem. Proc. Natl. Acad. Sci. USA 92: 2041-2045
  14. Sugahara, T., Grootenhuis, P. D. J., Sato, A., Kudo, M., Ben-Menahem, D., Pixley, M. R., Hsueh, A. J. W. and Boime, I. 1996. Expression of biologically active fusion genes encoding the FSH $\beta$ subunits: role of linker sequence. Mol. Cell Endocrinol. 125: 71-77
  15. Takeuchi, M., Inoue, N., Strickland, T. W., Kubota, M., Wada, M., Shimizu, R., Hoshi, S., Kozutsumi, H., Takasaki, S. and Kobata, A. 1989. Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinease hamster ovary cells. Proc. Natl. Acad. Sci. USA 86: 7819-7822
  16. Temin, H. M. 1989. Retrovirus variation and evolution. Genome 31: 17-22 https://doi.org/10.1139/g89-007