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Effects of Gamma-Irradiation on the Sterilization of Primordial Germ Cells in Quail

메추리 원시생식세포 감소를 위한 감마선 조사의 효과

  • Park, Kyung-Je (WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University) ;
  • Kim, Tae-Min (WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University) ;
  • Lee, Hyung-Chul (WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University) ;
  • Jang, Hyun-Jun (WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University) ;
  • Song, Gwon-Hwa (WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University) ;
  • Han, Jae-Yong (WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University)
  • 박경제 (서울대학교 농생명공학부 WCU 바이오모듈레이션) ;
  • 김태민 (서울대학교 농생명공학부 WCU 바이오모듈레이션) ;
  • 이형철 (서울대학교 농생명공학부 WCU 바이오모듈레이션) ;
  • 장현준 (서울대학교 농생명공학부 WCU 바이오모듈레이션) ;
  • 송권화 (서울대학교 농생명공학부 WCU 바이오모듈레이션) ;
  • 한재용 (서울대학교 농생명공학부 WCU 바이오모듈레이션)
  • Received : 2010.04.17
  • Accepted : 2010.05.26
  • Published : 2010.06.30

Abstract

Quail is a very useful animal model for studying vertebrate development because of its small body size and unique reproductive traits. This species is also ideal model for producing germline chimeras via transferring exogenous primordial germ cells (PGCs) into the recipient embryo. To increase the contribution efficiency of donor PGCs into recipients' tissues, decreasing the population of endogenous PGCs has been rate-limiting factor. We therefore conducted this study to investigate if gamma ($\gamma$)-irradiation depletes endogenous PGCs in developing quail embryo. Firstly, freshly laid stage X quail embryos were irradiated with various output of $\gamma$-irradiation and its teratogenic effect on the embryo was evaluated. Although a dose-dependent increase in the number of embryo showing malformation was found as the output increased (0, 250, 500, 750, and 1,000 rads), only a maximum of 10.1% of embryos were abnormal in 1,000 rads. Immunocytochemical analysis using the QCR1 antibody, which is specific marker for quail PGCs, was conducted to analyze the effect of sterilization. As results, $\gamma$-rays at a dose-rate of 500 rads/73 sec onto undeveloped stage X embryo significantly reduced the number of germ cells to an average of 75.55 % and 82.03 % in male and female embryos, respectively. We conclude that $\gamma$-ray selectively targets PGCs while affects minimally to the somatic development in quail embryo. Our results will not only provide important data for germline chimera production but can be used for analyzing the effect of ionized rays on the differentiating germ cells in various stages during animal development.

조류의 원시생식세포는 수용체 배자로의 주입을 통해서 생식선 카이메라 생산을 가능하게 하기 때문에 유전자 도입에 매우 효율적인 도구이다. 특히, 메추리는 성성숙이 빠르며 산란능력이 뛰어 나기 때문에 형질전환 조류 생산과 유전자 기능 연구에 매우 적합하다. 형질전환 조류 생산 효율을 높이기 위해서는 수용체 배자 내의 원시생식세포의 수를 줄이는 것이 필수적인 요소이지만, 아직까지 메추리에서 이러한 시도를 했다는 보고는 없다. 본 연구는 감마선 조사가 수용체 내의 원시생식세포의 수를 감소시킬 수 있는지 알아보았다. 먼저 0, 250, 500, 750, 1,000 rads 강도의 감마선을 갓 산란된 메추리알에 조사 후 5일령 배자에서 기형 발생빈도를 측정하였고, 0과 500 rads에서 17일째 부화율을 검정하였다. 그리고 500 rad의 감마선을 산란된 알에 73초간 조사 후 5일간 배양시킨 뒤 원시생식세포의 수를 측정하였다. 그결과, $1{\times}10^4$개의 세포 당 원시생식세포의 수는 수컷은 $107.75{\pm}3.96$에서 $80.30{\pm}4.34$로, 암컷에서 $99.56{\pm}3.22$에서 $81.67{\pm}3.72$로 원시생식세포수가 감소되었다. 이상의 연구 결과는 감마선 조사가 수용체 배자내의 원시생식세포를 감소시킬 수 있고, 이를 형질 전환 기술에 접목시켜서 생식선 카이메라 효율을 높일 수 있는 가능성을 보여 주고 있다.

Keywords

References

  1. Aigegil V, Simkiss K 1991 Sterilizing embryos for transgenic chimeras. Br Poult Sci 32:427-438. https://doi.org/10.1080/00071669108417368
  2. Aoyama H, Asamoto K, Nojyo Y, Kinutani M 1992 Monoclonal antibodies specific to quail embryo tissues: their epitopes in the developing quail embryo and their application to identification of quail cells in quail-chick chimeras. J Histochem Cytochem 40:1769-1777. https://doi.org/10.1177/40.11.1385517
  3. Carsience RS, Clark ME, Verrinder Gibbins AM, Etches RJ 1993 Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos. Development 117:669-675.
  4. Kagami H, Tagami T, Matsubara Y, Harumi T, Hanada H, Maruyama K, Sakurai M, Kuwana T, Naito M 1997 The developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mixed-sex germline chimeras to the offspring in the chicken. Mol Reprod Dev 48:501-510. https://doi.org/10.1002/(SICI)1098-2795(199712)48:4<501::AID-MRD11>3.0.CO;2-W
  5. Kim MA, Park TS, Kim JN, Park HJ, Lee YM, Ono T, Lim JM, Han JY 2005 Production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells into recipient embryos. Theriogenology 63:774-782. https://doi.org/10.1016/j.theriogenology.2004.04.014
  6. Lim JM, Kwon HM, Kim DK, Kim JN, Park TS, Ono T, Han JY 2006 Selective decrease of chick embryonic primordial germ cells in vivo and in vitro by soft X-ray irradiation. Anim Reprod Sci 95:67-74. https://doi.org/10.1016/j.anireprosci.2005.09.006
  7. Ogawa A, Solovei I, Hutchison N, Saitoh Y, Ikeda JE, Macgregor H, Mizuno S 1997 Molecular characterization and cytological mapping of a nonrepetitive DNA sequence region from the W chromosome of chicken and its use as a universal probe for sexing carinatae birds. Chromosome Res 5:93-101. https://doi.org/10.1023/A:1018461906913
  8. Padgett CA, Ivey WD 1959 Coturnix quail as a laboratory research animal. Science 129:267-268. https://doi.org/10.1126/science.129.3344.267
  9. Song Y, D'Costa S, Pardue SL, Petitte JN 2005 Production of germline chimeric chickens following the administration of a busulfan emulsion. Mol Reprod Dev 70:438-444. https://doi.org/10.1002/mrd.20218
  10. Tajima A, Naito M, Yasuda Y, Kuwana T 1993 Production of germ line chimera by transfer of primordial germ cells in the domestic chicken (Gallus domesticus). Theriogenology 40:509-519. https://doi.org/10.1016/0093-691X(93)90404-S
  11. Thoraval P, Lasserre F, Coudert F, Dambrine G 1994 Somatic and germline chimeras obtained from Brown and White Leghorns by transfer of early blastodermal cells. Poultry Sci 73:1897-1905. https://doi.org/10.3382/ps.0731897
  12. van de Lavoir MC, Diamond JH, Leighton PA, Mather-Love C, Heyer BS, Bradshaw R, Kerchner A, Hooi LT, Gessaro TM, Swanberg SE, Delany ME, Etches RJ 2006. Germline transmission of genetically modified primordial germ cells. Nature 441:766-769. https://doi.org/10.1038/nature04831

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