형질전환생쥐에서 Lck Promoter에 의한 Diphtheria Toxin-A Gene의 발현 분석

나루세겐지,이승현,최화식,이성호,박창식,진동일 한국동물생명공학회(구 한국동물번식학회) 2003 Reproductive & developmental biology Vol.27 No.3

본 연구는 생체 내 세포 및 조직배양기로서의 면역결핍동물을 개발할 목적으로 proximal lck promoter와 DT-A gene를 이용하여 형질전환생쥐을 생산하고 이 형질전환생쥐의 면역세포에서 DT-A gene이 발현되는지를 분석하였다. 형질전환생쥐와 정상생쥐로부터 thymus, spleen 및 liver에서 RNA를 추출하여 RT-PCR수행하였는데 정상생쥐의 조직에서는 어떠한 DT-A gene의 발현양상을 얻을 수 없었으나 형질전환생쥐의 thymus, spleen, liver에 DT gene의 발현을 확인할 수 있었고, Northern blotting을 이용하여 형질전환생쥐의 thymus, spleen 및 liver에서 DT-A gene이 강하게 발현되는 것으로 나타났다. 형질전환생쥐 F₁ 및 F₂ 산자의 혈액에서 T-cell 발달의 분포도를 확인하기 위해 CD4 및 CD8 ntibody를 이용하여 FACS analysis를 실시하였는데 형질전환생쥐의 혈액 내 mature T-cell인 single positive thymocyte의 수가 정상생쥐에 비해 감소하는 경향을 나타냈다. 정상생쥐의 혈액 내 T-cell 중 CD8/sup +/ T-cell의 경우 약 50%를 나타냈으나 형질전환생쥐의 경우 33%로 감소하였고, CD4/sup +/ T-cell은 정상생쥐에서 10%를 차지하고 있으나 형질전환생쥐에서는 5.9%로 감소되는 것으로 분석되었다. 그러므로 본 연구의 형질전환생쥐에서 lck promoter에 의해 초기 immature한 상태의 T-cell에서 DT-A gene이 발현되어 발육중인 T-cell이 파괴 되어 mature 상태인 CD4/sup +/ CD8/sup -/나 CD4/sup -/CD8/sup +/ cells (single-positive)들이 감소된 것으로 확인되었다. Transgenic mice containing Diphtheria Toxin-A (DT -A) gene fused to proximal lck promoter sequences was used for analysis of DT-A gene expression and thymocyte development. The diphtheria toxin gene was expressed in thymus, spleen and liver of transgenic mice confirmed by RT-PCR and Northern blotting. A FACS analysis with thymocyte cell surface antigens antibodies (CD4 and CD8) showed that the number of peripheral mature single positive thymocytes (CD4/sup +/ and CD8/sup +/ cells) T-cells was severely reduced in transgenic mice compared to that in the non-transgenic littermates. A relative portion of CD8/sup +/ single positive thymocytes was about 33.2% in transgenic peripheral T-cells while 50.6% in wild type. Reduction of CD4/sup +/ cell numbers in transgenic mice was observed (5.9% in transgenic versus 10.3% in non-transgenic). The data from analysis of these transgenic mice indicate that the proximal lck promoter regulated the expression of DT-A gene at high level in developing thymocytes and the DT-A disrupted developing thymocytes in transgenic mice.

형질전환생쥐에서 Lck Promoter에 의한 Diphtheria Toxin-A Gene의 발현 분석

나루세겐지,이승현,최화식,이성호,박창식,진동일 충남대학교 형질전환복제돼지연구센터 2004 논문집 Vol. No.8

Transgenic mice containing Diphtheria Toxin-A (DT-A) gene fused to proximal lck promoter sequences was used for analysis of DT-A gene expression and thymocyte development. The diphtheria toxin gene was expressed in thymus, spleen and liver of transgenic mice confirmed by RT-PCR and Northern blotting. A FACS analysis with thymocyte cell surface antigens antibodies (CD4 and CD8) showed that the number of peripheral mature single positive thymocytes (CD4^(+) and CD8^(+) cells) T-cells was severely reduced in transgneic mice compared to that in the non-transgenic littermates. A relative portion of CD8^(+) single positive thymocytes was about 33.2% in transgenic peripheral T-cells while 50.6% in wild type. Reduction of CD4^(+) cell numbers in transgenic mice was observed (5.9% in transgenic versus 10.3% in non-transgenic). The data from analysis of these transgenic mice indicate that the proximal lck promoter regulated the expression of DT-A gene at high level in developing thymocytes and the DT-A disrupted developing thymocytes in transgenic mice.

형질전환생쥐에서 1.7 kb 및 3.1 kb bovine -casein promoter가 human type II collagen 유전자의 발현조절에 관한 분석

나루세겐지,양정희,권혁빈,유승권,최윤재,박창식,진동일 한국발생생물학회 2003 한국발생생물학회 학술발표대회 Vol.2003 No.1

본 연구에서는 1.7kb 및 3.1kb bovine -casein promoter의 유전자 발현 조절능력을 알아보기 위해 1 7kb 및 3.1kb bovine -casein promoter에 human Type II Collagen 유전자를 연결해서 DNA microinjection으로 형질전환생쥐를 생산하였다. 총 8마리의 founder생쥐(1.7kb collagen : 5마리, 3.1kb collagen 3마리)를 생산하였고 이 founder생쥐와

면역결핍동물의 생산을 위한 형질전환생쥐의 분석

나루세겐지,양정희,이승현,최화식,이성호,박창식,진동일 한국동물생명공학회(구 한국동물번식학회) 2003 Reproductive & developmental biology Vol.27 No.2

본 연구는 생체 내 세포 및 조직배양기로서의 면역결핍동물을 개발할 목적으로 proximal Ick promoter와 DT-A유전자를 이용하여 형질전환생쥐를 생산하였고 형질전환생쥐의 면역기관에서 Di-phteria toxin이 발현되어 T-cell이 결핍되는지를 분석하였다. 총 암수 2마리의 형질전환생쥐를 PCR과 South-ern blotting으로 분석하여 얻었으며 이식유전자의 copy수는 약 2∼3 copy가 정착된 것으로 확인되었다. 형질전환생쥐의 thymus, spleen, liver 조직을 분리한 후 total RNA를 추출하여 poly(dT) primer 와 DT 특이적 primer를 이용하여 RT-PCR수행 결과 형질전환생쥐의 thymus, spleen, liver에서 DT gene이 발현되고 있는 것을 확인할 수 있었다. 형질전환생쥐의 이들 조직간에 DT 발현량에는 큰차이는 없는 것으로 확인되었다. 형질전환생쥐의 혈액에서 적혈구, 백혈구 ,혈소판, 헤모글로빈 등이 정상생쥐보다 감소되었고 특히 백혈구수와 혈소판의 수가 크게 감소되어 있는 것으로 나타났다. 또한 형질전환생쥐의 혈액을 CD3 antibody를 이용하여 FACS 분석을 실시하여 형질전환생쥐의 혈액 중 T-cell이 수가 비정상적으로 줄어든 것을 확인할 수 있었다. 본 연구에서는 Ick-DT 형질전환생쥐에서 DT유전자의 발현에 의한 T-cell 결핍을 유도할 수 있는 것으로 나타나 이를 바탕으로 돼지를 이용한 사람의 이종장기 배양용 형질전환동물을 생산하여 응용될 수 있을 것으로 사료된다. To determine whether the diphtheria toxin-A (DT) gene disrupts development of thymocytes in transgenic animal, the DT-A gene was used for the production of transgenic mice directed by proximal Ick promoter sequences. Two transgenic founder mice that contained several copies of transgene were produced by DNA microinjection and integration of transgene in transgenic mice was confirmed by PCR and Southern blotting analysis. Transgenic F₁ and F₂ mice were produced by outbreeding of founder and F₁ mice to investigate expression of transgene and phenotypes in transgneic mice. Expression of the diphtheria toxin gene was confirmed in thymus, spleen and liver of transgenic mice by RT-PCR. In circulating blood of transgenic mice, lower number of circulating white blood cells and platelets were observed compared with that of normal mice. In addition, transgneic mice had reduced number of circulating peripheral T-cells analyzed by FACS with anti-CD3 antibody. The data in these transgenic mice indicate that DT gene can play a disruptive role in developing thymocytes of transgenic mice resulted in lower number of T-cells that can be applicable to a wide range of tissues in other animals.

형질전환생쥐에서 1.7 kb 및 3.1 kb bovine -casein promoter가 human type II collagen 유전자의 발현조절에 관한 분석

나루세겐지,양전희,권혁빈,유승권,최윤재,박창식,진동일 한국수정란이식학회 2003 한국수정란이식학회 학술대회 Vol.2003 No.1

유산양 체세포를 이용한 돼지 난자의 이종간 핵이식 후 배발달에 관한 연구

장석민,나루세겐지,신영민,박창식,진동일 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10

This study was conducted to investigate the developmental ability of interspecies cloned embryos after nuclear transfer of goat fetal fibroblast cells into porcien cocytes. Recipient porcine anc goat oocytes were obtained from slaughterhouse and matured III vitro according to established protocols Enucleation was accomplished by aspirating the first polar body and cytoplasm and a single doner cell was individually microinjected into vitelline space of the enucleated oocyte. The reconstructed oocytes were electrically fused WIth O.3M mannitol fusion medium. After electro-fusion, interspecies reconstituted embryos were cultured in PZM-3 for 7 days. In porcine interspecies nuclear transfer With goat fetal fibroblast cells, the cleavage rate of reconstituted ernbryos were 58.9% which was no significant different from that in porcine nuclear transfer embryos (67.4%). However, the developmental rate into blastocyst stage was 5.4% in interspecies nuclear transfer which was significantly lower than that in porcine intraspecies nuclear transfer (13.6%). When the developmental ability of porcine interspecies nuclear transfer with goat cells was compared with goal intraspecies nuclear transfer. the cleavage rate of embryos were 59.2% and the developmental rate into morular and blastocyst stage was 13.6% in intespecies nuclear transfer which were significantly lower than those in intraspecies nuclear transfer embryos. This result indicated that porcine interspecies nuclear transfer with goat fetal fibrobiast cells showed the developmental potential in vitro with lower cleavage and developmental rate compared with intraspecies nuclear transfer.

유산양 체세포를 이용한 돼지 난자의 이종간 핵이식후 배발달에 관한 연구

장석민,나루세겐지,신영민,박창식,진동일 충남대학교 농업과학연구소 2006 농업과학연구 Vol.33 No.1

This study was conducted to investigate the developmental ability of interspecies cloned embryos after nuclear transfer of goat fetal fibroblast cells into porcien oocytes. Recipient porcine and goat oocytes were obtained from slaughterhouse and matured in vitro according to established protocols. Enucleation was accomplished by aspirating the first polar body and cytoplasm and a single donor cell was individually microinjected into vitelline space of the enucleated oocyte. The reconstructed oocytes were electrically fused with 0.3M mannitol fusion medium. After electro-fusion, interspecies reconstituted embryos were cultured in PZM-3 for 7 days. In porcine interspecies nuclear transfer with goat fetal fibroblast cells, the cleavage rate of reconstituted embryos were 58.9% which was no significant different from that in porcine nuclear transfer embryos (67.4%). However, the developmental rate into blastocyst stage was 5.4% in interspecies nuclear transfer which was significantly lower than that in porcine intraspecies nuclear transfer (13.6%). When the developmental ability of porcine interspecies nuclear transfer with goat cells was compared with goat intraspecies nuclear transfer, the cleavage rate of embryos were 59.2% and the developmental rate into morular and blastocyst stage was 13.6% in interspecies nuclear transfer which were significantly lower than those in intraspecies nuclear transfer embryos. This result indicated that porcine interspecies nuclear transfer with goat fetal fibroblast cells showed the developmental potential in vitro with lower cleavage and developmental rate compared with intraspecies nuclear transfer.

전기천공법과 Liposome 시약을 이용한 재래돼지 세포의 형질전환 효율비교

김백철,장석민,나루세겐지,박창식,진동일 한국동물생명공학회(구 한국동물번식학회) 2007 Reproductive & developmental biology Vol.31 No.2

PCR을 이용한 형질전환 유산양 체세포의 Transfection 효율 분석

신영민,장석민,나루세겐지,박창식,진동일 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10

PCR을 이용한 형질전환 유산양 체세포의 Transfection 효율 분석

신영민,장석민,나루세겐지,박창식,진동일 한국동물생명공학회(구 한국동물번식학회) 2006 Reproductive & developmental biology Vol.30 No.2