한국수정란이식학회지 (Journal of Embryo Transfer)

  • 제17권1호
  • /
  • Pages.45-53
  • /
  • 2002
  • /
  • 2508-755X(pISSN)
  • /
  • 2288-0178(eISSN)
한국수정란이식학회 (The Korean Society of Embryo Transfer)
권호 표지

외래유전자 도입정자를 이용한 돼지 체외성숙 난포란의 Intracytoplasmic Sperm Injection (ICSI) 후 후기 배로의 발달율과 외래유전자의 발현에 관한 연구

Development and Expression of Porcine Embryos by Direct Injection of Sperm Treated with Exogenous DNA

  • 정기화 (경상대학교 농업생명과학연구원) ;
  • 조성근 (경상대학교 농업생명과학연구원)
  • 발행 : 2002.04.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

본 연구는 sperm-mediated gene transfer를 이용하여 ICSI에 의한 형질전환동물 생산의 기초자료로서 활용하기 위해, ICSI에 사용될 정자의 조건과, 그에 따라 적합한 돼지 정자와 외래유전자의 전처리 및 ICSI를 통한 수정을 및 후기 배로의 발달율과 외래유전자의 발현 여부를 조사하여 다음과 같은 결과를 얻었다. ICSI에 이용될 정자의 조건에 따라 정소상체미부정자, 사출정자 및 동결정자를 이용하여 ICSI후 수정율은 각각 72.3%, 64.1% 및 74.1%로 나타나 유의적인 차이를 나타내지 않았으며, 또한 후기배로의 발달율에 있어서도 각각 17.6%, 18.7% 및 15.0%로 나타나 각 처리군간의 유의적인 차이는 나타나지 않았다. 그리고, ICSI후 전기적 자극을 실시한 군과 실시하지 않은 군에서 난자의 활성화에 따른 수정율은 대조구로 이용한 shame injection과 전기적 활성화를 실시하지 않은 군에서 각각 47.1%와 46.3%로 나타나 전기적 활성화를 실시한 군의 79.6%에 비해 유의적인 차이를 나타내었다. 후기배로의 발달율에 있어서도 전기적 활성화를 실시한 군에서는 24.1%로 나타나 전기 적 활성화를 실시하지 않은 군에서의 14.4%와 유의적인 차이를 나타내었다. 그리고 대조구로 이용한 shame injection에 있어서 후기 배로의 발달율은 2.5%로 낮은 결과를 나타내었다. 또한, 정자와 pcDNA LacZ유전자의 처리시 electroporation 방법을 실시하여 ICSI후 난자를 각각 전기적 활성화를 실시한 군과 실시하지 않은 군에 있어서 유전자 발현율은 각각 30.2%와 24.2% 나타났으나, 두 처리군간에 유의적인 차이는 나타나지 않았다. 그러나, 두 군에서 pcDNA LacZ 유전자는 모두 mosaic 발헌 양상을 보였다. 이상의 실험 결과들을 종합해 보면, ICSI에 사용될 수 있는 돼지의 정자는 정소상체미부정자, 사출정자 및 동결정자 모두가 이용 가능하며, ICSI후 추가 전기적 자극에 의한 난자의 활성화가 수정율과 후기 배로의 발달율을 향상시킬 수 있음을 시사하였다. 따라서, 돼지에 있어서 정자의 외래유전자 도입에 대한 정확하고 실용적인 방법은 보고되고 있지는 않은 상태로, 정자와 외래유전자의 처리법을 향상시키기 위하여 다양한 방법으로 많은 연구가 요구된다.

The main goal of this study was to produce transgenic porcine embryos by direct injection of sperm-mediated exogenous DNA. Spermatozoa (6$\times$10$^{6}$ sperms of final concentration) were mixed with pcDNA LAC Z (20 ng/$\mu$l) and subjected into electroporation (300~750 volts, 25 $\mu$F, 0.4 cm electrode). After sperm injection, the oocytes were activated electrically (1.7 KV/cm, 30$\mu$sec, single pulse) in 0.3 M mannitol solution or not. The sperm injected eggs were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air fur 144 h. The rates of cleavage and development into blastocyst stage in activation group were significantly higher than those of non-activation group (79.6% and 24.1% vs. 46.3% and 14.4%, respectively, p<0.05). Control oocytes and shame injection were developed to blastocysts low (2.5%). Sixty five (27.1%) out of 240 embryos observed in activation and non-activation groups were showed positive by X-gal staining. However, all embryos in both groups were expressed partial or mosaic pattern. These results suggested that electrical stimulation far oocytes activation after sperm injection enhances the incidence of both fertilization and development fellowing sperm injection in the pig. Our study also suggested that sperm-mediated transfer of exogenous DNA by ICSI would be used as a valuable tool for the production of transgenic porcine embryos.

키워드

참고문헌

  1. Bachiller D, Schellander K, Peli J and Ruther U. 1991. Liposome-mediated DNA uptake by sperm cells. Mol. Reprod. Dev., 30:194-200 https://doi.org/10.1002/mrd.1080300305
  2. Brackett BG, Baranska W, Sawicki W and Koprowski H. 1971. Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc. Natl. Acad. Sci., USA. 68(2):352-357
  3. Chan AWS, Luetjens CM, Dominko T, Ramalho-antos J, Simerly C, Hewitson L and Schatten G. 2000a. TransgenelCSI review: foreign DNA transmission by intracytoplasmic sperm injection in Rhesus monkey. Mol. Reprod. Dev., 56:325-328 https://doi.org/10.1002/(SICI)1098-2795(200006)56:2+<325::AID-MRD25>3.0.CO;2-N
  4. Chan, AWS, Luetjens CM, Dominko T, Ramalho-Santos J, Simerly C, Hewitson L and Schatten G. 2000b. Foreign DNA transmission by ISI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP exepression and live Rhesus monkey birth. Mol. Human Reprod., 6:26-33 https://doi.org/10.1093/molehr/6.1.26
  5. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Leon PFA 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
  6. Di Berardino D, Gil MA, Parrilla I, Fernandez MA, Coppola G, Mazza MR, RToca J, Vazquez JM, Lucas X and Martinez EA. 2000. Pronuclear formation and embryo development in pig oocytes fertilized by intracytoplasmic sperm injection. Theriogenology, 53(1):389. Abstr
  7. Gagne, MB, Pothier F and Sirard MA. 1991. Elec-troporation of bovine spermatozoa to carry foreign DNA in oocytes. Mol. Reprod. Dev., 29:6-15 https://doi.org/10.1002/mrd.1080290103
  8. Hammer RE, Pursel VG, Rexroad CE, Wall Jr RJ, Bolt DJ, Ebert KM, Palmiter RD and Brinster RL. 1985. Production of transgenic rabbits, sheep and pigs by microinjection. Nature, 315: 680-683 https://doi.org/10.1038/315680a0
  9. Horan R, Powell R, Bird JM, Gannon F and Houghton JA. 1992. Effects of electropermea-bilizatio on the association of foreign DNA with pig sperm. Arch. Androl., 28:105-114 https://doi.org/10.3109/01485019208987687
  10. Huguet E and Esponda P. 1998. foreign DNA introduced into the vas deferens is gained by mammalian spermatozoa. Mol. Reprod. Dev., 51:42-52 https://doi.org/10.1002/(SICI)1098-2795(199809)51:1<42::AID-MRD5>3.0.CO;2-W
  11. Jolliff WJ and Prather RS. 1997. Parthenogenetic development of in vitro-matured, in vitro-cultured porcine oocytes beyond blastocyst. Biol. Reprod., 56:544-548 https://doi.org/10.1095/biolreprod56.2.544
  12. Kikuchi K, Izaike Y, Noguchi J, Furukawa T, Daen FP, Niato K and Toyoda Y. 1995. Decrease of histon Hl kinase activity in relation to parthenogenetic activation of pig follicular oocytes matured and aged in vitro. J Reprod. Fertil., 105:325-330 https://doi.org/10.1530/jrf.0.1050325
  13. Kim JH, Jung-Ha HS, Lee HT and Chung KS. 1997. Development of a positive method for male stem cell-mediated gene transfer in mouse and pig. Mol. Reprod. Dev., 46:1-12 https://doi.org/10.1002/(SICI)1098-2795(199701)46:1<1::AID-MRD1>3.0.CO;2-V
  14. Kim NH, Lee JW, Jun SH, Lee HT and Chung KS. 1998. fertilization of porcine oocytes following intracytoplasmic spermatozoon or isolated sperm head injection. Mol. Reprod. Dev., 51:436-444 https://doi.org/10.1002/(SICI)1098-2795(199812)51:4<436::AID-MRD11>3.0.CO;2-Q
  15. Kim NH, Shin JS, Kim C, Jun SH, Lee HT and Chung K-S. 1999. Fertilization and in vitro development of porcine oocytes following intracytoplasmic injection of round spermatid or round spermatid nuclei. Theriogunology, 51:1441-1449 https://doi.org/10.1016/S0093-691X(99)00088-6
  16. Kim T, Leibfried-Rutledge ML and Fiist NL. 1993. Gene transfer in bovine blastocysts using replication with Gibbon ape leukemia virus envelopes. Mol. Reprod. Dev., 35:105-113 https://doi.org/10.1002/mrd.1080350202
  17. Kolbe T and Holtz W. 1999. Intracytoplasmic injection (ICSI) of in vivo or in vitro matured oocytes with fresh ejaculated or frozen-thawing epididymal spermatozoa and additional calcium-ionophore activation in the pig. Theriogenology, 52:671-682 https://doi.org/10.1016/S0093-691X(99)00161-2
  18. Kolbe T and Holtz W. 2000. Birth of a piglet derived from an oocyte fertilized by intracytoplasmic sperm injection (ICSI). Animal Reproduction Science, 64:97-1001 https://doi.org/10.1016/S0378-4320(00)00204-9
  19. Kure-bayashi S, Miyake M, Katayama M, Miyano T and Kato S. 1996. Development of porcine blastocysts from in vitro-matured and activated haploid and diploid oocytes. Theriogenology, 46:1027-1036 https://doi.org/10.1016/S0093-691X(96)00267-1
  20. Kuretake S, Kimura Y, Hoshi K and Yanagimachi R. 1996. Fertilization and development of mouse oocytes injected with isolated sperm heads. Biol. Reprod., 55:789-795 https://doi.org/10.1095/biolreprod55.4.789
  21. Labitrano M, Camaioni A, Frati VM, Dolci S, Farace MG and Spadafora C. 1989. Spermcells as vectors for introducing foreign DNA into eggs: genetic transformation of mice. Cell, 57:717-723 https://doi.org/10.1016/0092-8674(89)90787-3
  22. Leal CL and Liu L. 1998. Differential effects of kinase inhibotor and electrical stimulus on activation and histon Hl kinase activity in pig oocytes. Anim. Reprod. Sci., 52:51-61 https://doi.org/10.1016/S0378-4320(98)00084-0
  23. Lee JW, Jeong BS, Riesen J, Hoagland T and Yang X. 2001. Fertilization and embryonic development following injection of frozen-thawing spermatozoon into in vitro matured porcine oocyte. Theriogenology, 55(1):506. Abstr
  24. Liu L and Moor RM. 1997. Factors affecting electrical activation of porcine oocyte matured in vitro. Anim. Reprod. Sci., 48:67-80 https://doi.org/10.1016/S0378-4320(97)00044-4
  25. Martin MJ. 2000. Development of in vivo-matured porcine oocytes following intracytoplasmic sperm injection. Biol. Repord., 63:109-112 https://doi.org/10.1095/biolreprod63.1.109
  26. Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H and Perry ACF. 2000. Pig cloning by microinjection of fetal fibroblast nuclei. Science, 1188-1190
  27. Parrington J, Swann K, Schevchenko VI, Sesay AK and Lai FA. 1996. Calcium oscillations in mammalian egga triggered by a soluble sperm protein. Nature, 379:364-368 https://doi.org/10.1038/379364a0
  28. Perry AC, Wakayama T, Kishikawa H, Kasai T, Okabe M, Toyoda Y and Yanagimachi R. 1999. Mammalian transgenesis by intracytoplasmic sperm injection. Science, 14:1180-1183
  29. Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares DL, Colman A and Campbell KH. 2000. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature, 407:86-90 https://doi.org/10.1038/35024082
  30. Prather RS, Boice ML, Gibson J, Hoffman KE and Parry TW. 1995. In vitro development of embryos from sinclair miniature pigs: A preliminary report. Theriogenology, 43:1001-1007 https://doi.org/10.1016/0093-691X(95)00064-F
  31. Rieth A, Pothier F and Sirars MA. 2000. Electroporation of bovine spermatozoa to carry DNA containing highly repetitive sequences into oocytes and detection of homologous recombination events. Mol. Reprod. Dev., 57:338-345 https://doi.org/10.1002/1098-2795(200012)57:4<338::AID-MRD5>3.0.CO;2-K
  32. Robl MJ, Collas P, Fissore R and Dobrinsky J. 1992. Electrically induced fusion and activation in nuclear transplant embryos, In: Guide to electroporation and electrofusion. New York: Academic Press:535-551
  33. Schnieke AE, Kind AJ, Ritchie WA, Mycook K, Scott AR, Retchie 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
  34. Sim SW, Kim YH, Jun SH, Lim JM, Chung HM, Ko JJ, Lee HT, Chung KS and Shim H. 2000 Transgenesis of porcine embryos using intracytoplasmic sperm injection. Theriogenology, 53(1):521. Abstr
  35. Spadafora C. 1998. Sperm cells and foreign DNA: a controversial relation. Bioessays., 20:955-964 https://doi.org/10.1002/(SICI)1521-1878(199811)20:11<955::AID-BIES11>3.0.CO;2-8
  36. Tsukui T, Kanegae Y, Saito I and Toyoda Y. 1996.Transgenesis by adenovirus-mediated gene transfer into mouse zona-free eggs. Nature Biotechnol., 14:982-985 https://doi.org/10.1038/nbt0896-982
  37. Wakayama T, Whittingham DG and Yanagimachi R. 1998. Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection. J. Reprod. Fertil., 112:11-17 https://doi.org/10.1530/jrf.0.1120011
  38. Wakayama T and Yanagimachi R. 1998. Develop-ment of normal mice from oocytes injected with freeze-dried spermatozoa. Nat. Biotechnol., 16:639-641 https://doi.org/10.1038/nbt0798-639
  39. Wang WH, Abeydeera LR and Prather RS. 1998. Functional analysis of activation of porcine oocytes by spermatozoa, calcium ionophore, and electrical pulses. Mol. Rerod. Dev., 51:346-353 https://doi.org/10.1002/(SICI)1098-2795(199811)51:3<346::AID-MRD15>3.0.CO;2-0
  40. Yamauchi Y, Sasada H, Sugawgra S and Nagai T. 1996. Effect of culture conditions on arificial activation of porcine oocytes matured in vitro. Reprod. Fertil. Dev., 8:1153-1156 https://doi.org/10.1071/RD9961153
  41. Zhu J, Telfer EE, Fletcher J, Springbett A, Dobrinsky JR, De Sousa PA and Wilmut I. 2002. Improvement of an electrical activation protocol for porcine oocytes. Biol. Reprod., 66:635-641 https://doi.org/10.1095/biolreprod66.3.635