• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.36-36
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• 2002

This study was designed to examine the ability of the bovine (MII) oocytes cytoplasm to support several mitotic cell cycles under the direction of differentiated somatic cell nuclei of bovine, human, porcine and mouse. Bovine GV oocytes were matured in TCM-l99 supplemented with l0% FBS. At 22 h after IVM, denuded recipient oocytes were stained with 5 ㎍/㎖ Hoechst and their 1 st polar body (PB) and MII plate were removed by enucleation micropipette under. (omitted)

• 한국가축번식학회지
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• 제23권4호
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• pp.393-398
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• 1999

Somatic cell nuclear transfer is an efficient technique for the multiplication of elite livestock, engineering of transgenic animals, cell therapy and xenotransplantation, and analyzing the interactions between nucleus and cytoplasm, for various agricultural, biomedical and research purposes. Since the first somatic cell clone lamb was born, tremendous progress has been made toward developing technology for animal cloning. Viable farm animals and mice have now been produced by nuclear transfer using various fetal and adult somatic cells as nuclei donors. Transgenic clones were also produced from nuclear transfer of transfected somatic cells. In the future, somatic cell nuclear transfer will provide more numerous opportunities, both in basic and appled research as well as immediate uses in the generations of superior clone and transgenic animals. However, further technology refinement and improved understanding of the process are essential for commercial and basic research applications.

• Applied Microscopy
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• 제18권1호
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• pp.1-20
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• 1988

There's been arguments on the different morphological status between the nucleus accumbens septi and nucleus fundus striati of ventral striatum. Authors carried out the comparative study on the neuronal cell types of these nuclei, in the chick and the rat. Results are summarized as follows: In the nucleus accumbens septi of the chick, there found 3 main cell types. Type I cells are oval or spindle-shaped. They are the most abundant cell types, comprising more than 80% of neurons. The pale nucleus is usually indented. The cytoplasm is also pale and contains small amount of mitochondria, rough r-ER and Golgi complexes. This cell has a few symmetric synapses on the cell membrane. Type II cells are pale large cells. They are polygonal or irregularly-shaped. They contain pale spherical nucleus, and the pale cytoplasm with relatively large amount of mitochondria, free ribosomes and well-developed Golgi complexes. Some axo-somatic synapes are found on the cell. Type III cells are oval or spherical-shaped. The nucleus is relatively pale and large, In the dense cytoplasm, well developed. r-ER formed typical Nissl's body, and there found many mitochondria, ribosomes and lysosomes. In the chick fundus striati nucleus, there also found 3 main cell types. Type I cells are small and spindle-shaped. This type is the most abundant one and constitutes more the 80% of the neurons. Morphological features other than it's shape, is generally similar with that of Type I cell in the nucleus accumbens. Type II cells are irregularly shaped large cells. Dense cytoplasm contains large amount of cell organelles. Some axo-somatic synapses are found. Type III cells are small dense cells. This oval cell contains the oval nucleus, and the plentiful cytoplasm with well developed r-ER, ribosomes and mitochondria. In the nucleus accumbens septi of the rat, there found 4 main cell types. Type I cells are small, oval or spherical cells, comprising more than 90% of all the neurons. Spherical nucleus shows typical chromatin rim along the nuclear membrane. Dense cytoplasm contains many ribosomes and mitochondria. Type II cells are large oval cells. The eccentric nucleus is deeply invaginated. Pale cytoplasm contains large amount of ribosomes, Golgi complexes, mitochondria, and dense bodies. Type III cells are pale, large, oval cells. They contain moderate amount of ribosomes and mitochondria, and some scattered stacks of r-ER. Type IV cells are small pale cells. Small oval nucleus is indented and shows chromatin rim. Only small amount of ribosomes and mitochondria can be found. In the nucleus fundus striati of the rat, there also found 4 main cell types. Type I cells are spherical or oval cells, comprising more than 90% of the neurons. The chromatin rim of the spherical nucleus is not so prominent as compared to the rim of type I cell in the nucleus accumbens septi. The cytoplasm contains moderate amount of mitochondria, ribosomes and some scattered r-ER. A few axo-somatic synapses were found. Type II cells are small round or polygonal cells. Golgi complexes are especially well-developed in this cell type. The cytoplasm also contains moderate amount of mitochondria, ribosomes, and dense bodies. Type III cells are small cells. The large nucleus shows prominent chromatin rim. The cytoplasm contains many ribosomes and mitochondria. Type IV cells are large, spheircal or oval cells. The nucleus is deeply indented. The plentiful cytoplasm contains large amount of ribosomes, mitochondria, Golgi complexes, neurotubules, but not r-ER. In the present study, it is clear that the nucleus accumbens septi and the nucleus fundus striati are independant cell groups, according to their cytoarchitectonics and the ultrastructural features of their cell types.

• Asian-Australasian Journal of Animal Sciences
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• 제21권11호
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• pp.1665-1672
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• 2008

Interspecies somatic cell nuclear transfer (iSCNT) is a useful method to preserve endangered species and to study the reprogramming event of a nuclear donor cell by the oocyte. Although several studies of iSCNT using murine cells and bovine oocytes have been reported, the development of murine-bovine iSCNT embryos beyond the 8-cell stage has not been successful. In this paper, we examined the developmental potential of embryos reconstructed with a murine embryonic fibroblast as the nuclear donor and a bovine oocyte as the cytoplasm recipient. The reconstructed embryos were cultured in CZB (murine medium) or CR1aa (bovine medium). In addition, for the development of a murine-bovine iSCNT blastocyst, the antioxidant ${\beta}$-mercaptoethanol (${\beta}ME$) was supplemented to CR1aa medium. Furthermore, to verify the mouse genome activation in murine-bovine iSCNT embryos, RT-PCR analysis of murine Xist was performed. The development of the murine-bovine iSCNT embryos cultured in CR1aa was significantly higher than that in CZB (p<0.05). With respect to the effect of BME on the development of the murine-bovine iSCNT blastocyst, addition of BME produced a significant increase in blastocyst development (p<0.05). Karyotype analysis confirmed that the reconstructed embryos were derived from murine cells (40XX). The Xist gene was gradually increased from the 8-cell stage to the blastocyst stage. This is the first report of blastocyst development of iSCNT embryos derived from murine somatic cells and bovine oocytes. These results demonstrate that bovine cytoplasm can support the development of later stages of a preimplantation embryo from murine-bovine iSCNT.

• Asian-Australasian Journal of Animal Sciences
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• 제24권1호
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• pp.1-8
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• 2011

Large quantities of high-quality recipient oocytes with uniform cytoplasm are needed for research in the promising field of somatic cell nuclear transfer (SCNT) and embryonic stem cell research. In canines, however, it is difficult to obtain large quantities of oocytes because each donor produces a limited number of mature oocytes in vivo. Although in vitro maturation (IVM) is considered an alternative approach to oocyte production, this technique is still too rudimentary to be used for the production of highquality, uniform oocytes in large quantities. One technique for overcoming this difficulty is to use oocytes obtained from different species. This technique is known as interspecies SCNT (iSCNT). This review provides an overview of recent advances in canine - porcine interspecies SCNT.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2002년도 제42차 춘계학술대회
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• pp.87.1-87.1
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• 2002

• 한국가축번식학회지
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• 제26권3호
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• pp.235-243
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• 2002

본 연구는 몇 몇 포유류로부터 얻은 공여세포핵의 proliferation을 돕기 위한 수핵난자로 소 난자의 세포질을 사용하였으며, 공통 세포질로써의 능력을 시험하였다. 소 난자와 소, 사람, 돼지, 생쥐의 체세포와의 결합에서 유래한 각 종별 핵이식란의 융합율, 분할율, 배발달률 그리고 염색체 수를 조사하였다. 1. 소, 돼지, 생쥐, 사람 각각의 체세포를 이용한 핵이식란의 융합율은 70.2% 70.2% 72.4%와 63.0 %로 유의차를 보이지 않았다. 2. 소, 돼지, 생쥐, 사람 각각의 체세포를 이용한 핵이식란의 체외분할 ($\geq$2cell cleavage)율 또한 60.6%, 63.7%, 54.1%와 62.7%로 유의차가 없었다. 3. 각 종별로 체외분할이 일어난 시기를 조사한 결과 종에 관계없이 대체로 활성화 처리 후 24시간째에 대부분 일어나는 것을 볼 수 있었다. 그러나 점차적으로 분할이 계속 이루어지면서 발달시기가 종 특이적인 특성을 나타내는 것을 볼 수 있었다. 4. 이종간 핵이식의 후기 배발달 (상실배와 배반포) 률을 살펴보면 소와 사람의 체세포를 사용했을 경우 17.5%, 4.3%의 결과를 나타내었으며, 돼지와 생쥐의 체세포를 사용한 경우 16세 포기 이후 단계에 발달중지 현상을 볼 수 있었다. 5. 4~8 세포기 정도의 각 종별 핵이식란 할구를 분석에 사용하였을 때, 공여핵으로 사용된 종의 염색체 수와 일치하는 결과를 볼 수 있었다. 이상의 결과를 종합해 볼 때 성숙한 소 난자의 세포질은 소뿐만 아니라 돼지, 생쥐, 사람과 같은 다양한 포유류의 핵을 사용하여 핵이식을 하였을 때에도 발달이 가능하다는 것을 보여주고 있다.

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.109-113
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• 2007

This study was conducted to evaluate the effect of cytochalasin B (CB) treatment in the activation medium on the development of somatic cell nuclear transfer (SCNT) rat embryos. Fetal fibroblast cells were isolated from a Day 14.5 fetus, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM $SrCl_2$ with or without CB for 4 hr, and formation of pseudo-pronucleus (PPN) was checked at 18 hr after activation. Then, they were transferred into day 1 pseudopregnant recipients (Hooded Wistar) or cultured for 5 days to check their developmental competence in vivo or in vitro. The number of PPN was not affected by CB treatment during the activation. However, CB treatment supported pre-implantation development of rat SCNT embryos. Embryos generated by the procedures of SCNT were also capable of implanting, with 1 implantation scar found from a recipient following the transfer of 87 SCNT embryos to four foster mothers. The result of the present study shows that rat SCNT embryo can develop to post-implantation stage following treatment with CB.

• 한국수정란이식학회지
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• 제15권1호
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• pp.23-31
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• 2000

This study was conducted to investigate the effects of quiescent treatment of donor cells and activation treatment time of recipient cytoplasm on nuclear remodeling and in vitro development of somatic cell-cloned bovine embryos. Serum starved, confluent and nonquiescent cycling adult skin cells were teansferred into enucleated oocytes. Nuclear transfer oocytes were activated at 30 min, 1 and 2 hrs after electrofusion. Some nuclear transfer embryos(23% to 35%) extruded a polar body, which was not affected by quiescent treatment of donor cells and activiation time of recipient cytoplasm. About 68% of nuclear transfer embryos fused with a serum starved cells has a chromatin clump, but which was not different from embryos fused with confluent(51%) and nonquiescent(47%) cells. The proportion of embryos with a single chromatin clump was sightly increased when nuclear transfer embryos were activated within 30 min after fusion(69%) compared to those were activated at 1 and 2 hrs after fusion, but there was not significantly different. Development rates to the blastocyst stage were 8.6% and 15.9% when serum starved and confluent cells were transferred, which were higher than that of control group. Developmental rate to the blastocyst stage was higher in embryos were activated within 30 min after fusion (17.3%) compared to those of embryos were activated at 1 and 2 hrs after fusion (P<0.05). From the present result, it is suggested that quiescent treatment of donor cells and activation time of recipient cytoplasm can affect the in vitro development. Quiescent plasm activation within 30 min after fusion could increase the number of embryos with a normal chromation structure, which results in increased in vitro development.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.66-66
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• 2001

Since the first birth of pig derived from embryonic cells by nuclear transfer, many researches to produce cloned pig have been carried out. Recently, two reports about the birth of somatic cell cloned pigs using in vivo oocytes and also Betthauser et al. (2000) reported the birth of somatic cell cloned pigs using in vitro oocytes. So here we investigated the effect of activation method and culture medium on in vitro development of porcine nuclear transfer embryo using fetal fibroblast. Oocytes derived from slaughter house obtained ovaries were matured for 42 to 44 h in TCM 199. Matured oocytes were denuded using 0.1% hyaluronidase and then Oocytes with the first polar body were used for enucleation by aspirating the first polar body and adjacent cytoplasm in TCM 199 supplemented with 7.5 $\mu\textrm{g}$ cytochalasin B. Petal fibroblast cells were prepared from 35 days old fetus. To be used as donor cells, fetal fibroblast cells were serum starved for 3 to 5 days and then isolated into single co:1 by trypsinization. Nuclear transfer embryos were fused using 2 times 1.25㎸ for 30$mutextrm{s}$. Fused NT embryos were activated with calcium ionophore (CI) and 6-dimethyl-aminopurine (6-DMAP). Activated oocytes were cultured in NCSU 23 or BECM 3 for 6 days. There was no significant difference between chemical activation and no chemical activation for blastocyst development rate(11.6 vs. 14.8%). However, cell number was significantly higher when NT embryos were activated with CI and 6-DMAP (31.2 vs. 22.6). When NT embryos were cultured in NCSU 23 or BECM 3, blastocyst development rate was 16.4 and 13.2%, respectively, and cell number was 31.5 and 24.1, respectively. These results suggest that chemical activation after fusion and culture in NCSU 23 could increase cell number of porcine NT embryos.