[논문]Demecolcine-assisted Enucleation에 의한 소 형질 전환 핵이식란 생산

조종기; 손정민; 이두수; 윤기영; 신상태

Demecolcine-assisted Enucleation에 의한 소 형질 전환 핵이식란 생산
Production of Bovine Transgenic Somatic Cell Nuclear Transfer Embryos by Demecolcine-assisted Enucleation 원문보기

韓國受精卵移植學會誌 = Korean journal of embryo transfer, v.21 no.2, 2006년, pp.85 - 93

조종기 (충남대학교 수의과대학) , 손정민 (충남대학교 수의과대학) , 이두수 (충남대학교 수의과대학) , 윤기영 () , 신상태 (충남대학교 수의과대학)

초록
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본 연구는 소 형질 전환 체세포 핵이식에서 용이한 탈핵을 위해 demecolcine을 이용할 시 탈핵율과 핵이식란의 발육능을 높이기 위한 최적의 조건을 알아보고자 실시되었다. 도축장 유래 미성숙 난자를 18시간 체외성숙 후 제1극체가 확인된 성숙 난자를 0.1, 0.2, 0.4 및 0.8 ug/ml의 demecolcine이 첨가된 배지에서 1시간 더 처리한 다음 세포막이 돌출되어 있는 난자를 체세포 핵이식에 공여하여 각 군간 배반포로의 발육능을 비교하였다. Demecolcine 처리 후 핵이 포함된 셰포막의 protrusion rates를 각 군간 비교한 결과 0.2, 0.4 및 0.8 ug/ml 군에서 0.1 ug/ml 군보다 유의적으로 높았으며 (82.8, 86.2, 90.4 vs. 70.1%), conventional blind 방법과 비교한 결과 demecolcine를 이용한 군에서 유의적으로 높은 탈핵율을 보였다(75.3 vs. 96.2%; p<0.05). 체세포 핵이식란의 발육능 비교에서는 0.1 및 0.2ug/ml 군에서 대조군과 함께 유의적으로 높은 분할율 및 배반포로의 발육능을 보였다(p<0.05). 결론적으로 소 형질전환 체세포 핵이식을 위한 탈핵시 높은 탈핵율과 배반포로의 발육율을 얻을 수 있는 demecolcine의 적정농도는 0.2ug/ml이라고 사료된다.

주제어

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가설 설정

3. The fusion rates were not significantly different between groups regardless of demecolcine concentration.

제안 방법

8 ug/ ml). In the second experiment, comparison of enucleation rates between blind-enucleation and chemical-assisted group was conducted. In the third experiment, developmental potential of bovine transgenic SCNT embryos using chemically treated enucleation was compared with untreated control embryos.
8% (w/v) BSA and cultured at 39℃, 5% CO? atmosphere. The reconstructed embryos were cultured for 7 days after fusion, and cleavage and blastocyst formation were monitored under a stereomicroscope and GFP expression were confirmed under FITC filter set (Fig. 3).

성능/효과

2. The enucleation rates of oocytes using the demecolcine-assisted method were significantly higher than that by the conventional blind method.
In conclusion, an efficient and reliable chemically enucleation method for bovine oocytes has been suggested in this study by increasing the enucleation rates and development competence in the 0.2 ug/ml demecolcine groups. Further studies to investigate the effect of demecolcine on developmental ability of oocytes are also required to increase the efficiency of bovine SCNT.
In the experiment 2, the enucleation rate using the demecolcine-assisted method was significantly higher than that by the conventional blind method (Table 2).
, 1998), abnormal MPF concentrations by demecolcine treatment might affect the development competences. In the experiment 3, although significantly lower development rates were shown in the high concentration groups (0.4 and 0.8 ug/ml), higher development competences were achieved in the low concentration groups (0.1 and 0.2 ug/ml) similar to control group. However, we could not achieve relatively high rates of membrane protrusion containing chromatin after treatment of 0.
In the experiment 3, the proportions of transgenic SCNT embryos that fiised were not significantly different between control and groups treated with demecolcine at the concentration of 0.1, 0.2, 0.4 and 0.8 ug/ml (Table 3). However, in the proportions of cleavage and developed to blastocyst, there were significant differences between groups (Table 3).

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