• Reproductive and Developmental Biology
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• 제28권4호
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• pp.241-245
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• 2004

This study was conducted to investigate nuclear remodeling and developmental rate following nuclear transfer of fetal fibroblast cells, ear skin cells and oviduct epithelial cells into porcine recipient oocytes. To test par-thenogenetic activation, oocytes were treated with a 6-dimethylaminopurine (6-DMAP), a single DC-pulse (DC), calcium ionomycin (ionomycin), DC+6-DMAP and ionomycin + 6-DMAP after in vitro maturation. For nuclear transfer, in vitro matured oocytes were enucleated, and donor cells were transferred into oocytes. Cloned embryos were fused and stimulated with 6-DMAP for 4 h and cultured in vitro for 6 days. Among treatments for parthenogenesis, the activation rate of DC +6-DMAP treatment was significantly higher than that of single treatment roups (p<0.01), except for DC treatment group. However, the difference was not significant in activation rate compared to other complex treatment groups. Nuclear swelling of the cloned embryos was initiated at 60 min after stimulation and increased afterwards. Fusion rates were not different among different donor cells. Cleavage rates of DC treatment groups were significantly higher than those of DC+6-DMAP treatment groups (p<0.05) in case that fetal fibroblast and ear cells were used for nuclear donor. The cloned embryos from developed to blastocysts in oviduct epithelial cell nuclear transfer with DC+6-DMAP treatment was significantly higher compared to those with DC only treatment (p<0.05). However, no blastocyst was developed from nuclear transfer of fetal fibroblast and ear cells regardless of activation treatments. Based on these results, a proper activation stimulation may be necessary to increase the activation rate and the development to blastocyst in cloned porcine embryos.

• 한국가축번식학회지
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• 제24권2호
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• pp.217-222
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• 2000

본 연구는 세포의 휴면처리가 소 태아섬유아세포 유래 핵이식란의 핵형변화와 체외발육에 미치는 영향을 검토하였다. 임신 3∼4 개월령 한우 웅성 태아의 피부세포를 채취하여 계대배양 후 동결하였다가 핵이식 전에 혈청기아처리 또는 confluency방법으로 휴면처리를 하여 미수정란의 탈핵세포질에 이식하였다. 전기융합과 활성화처리 후 7∼9 일간 체외배양하여 발육능을 검토하였으며, 일부는 whole-mount 법으로 고정하여 염색질 구조를 관찰하였다. 복제란의 극체방출율은 혈청기아처리구와 confluence 구에서 각각 24.5% 와 20.3% 로 무처리구(36.0%) 에 비해 낮은 경향을 보였다. 활성화 후 1개의 염색질괴를 갖는 복제란은 혈청기아처리구(50.9%)와 confluence구 (49.2%)가 무처리구 (40.0%) 보다 높은 경향을 보였다. 극체방출에 따른 염색질의 구조는 극체 미방출구에서 정상적인 1개의 염색질괴를 갖는 핵이식란이 60.5% 로, 극체 방출구 (4.7%) 에 비하여 유의적으로 높았다 (P＜0.01). 배반포기 발육율은 혈청기아처리구 (21.7%) 와 confluence (20.9%) 가 무처리구 (14.1%) 에 비하여 비교적 높게 나타났다. 본 연구의 결과 혈청기아처리나 confluency 방법에 의한 donor 세포의 휴면처리는 태아섬유아세포 유래 핵이식란의 비정상적인 핵형변화를 감소시켜 체외발육능을 향상시키는 것으로 사료된다.

• 한국가축번식학회지
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• 제17권2호
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• pp.113-120
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• 1993

본 연구는 제외생산된 돼지 수정란의 처1외발생율을 제고하기 위하여 각종 배양액파 돼지난구세포 혹은 생 쥐태아간세포와의 공동배양 효과플 조사하였다 m-KRB, BECM 및 TCM-HEPES 배양액을 공시하 여 제외수정란을 배양한 결과 배반포기까지 발달하는 비율은 전처리구에서 0~1.0%로써 극히 저조하였다. 특히 대부분의 수정란은 4-세포기 단계에서 발달이 정지되었다. 한편, 단층세포가 유도된 돼지 난구세포나 생쥐 태아간세포와 함께 제외수정란을 공동배양한 결파 2, 4-, 8~16-, 32-세포기, 상실배가 빛 배반포로 받달하는 비율은 각각 61.1~67.0%, 59.0~58.0%, 42.5~43.1%, 28.4~30.2% 및 20.4~21.0%였다 이러한 결파는 단순배양액에서 체외배양한 수정란의 발탄 성적 보다유의하게 높은 것이었다. 이상의 결과를 종합하여 볼 때 1세포기 수정란을 체외에서 배양할때 체세포와의 공동배양은 수정란의 체외발달을 촉진하는 것으로 생각된다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.11-11
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• 2002

Interspecies nuclear transfer has been interested to determine ability of oocyte cytoplasm to support reprogramming of somatic cell nuclei of different species. In this study, we investigated developmental ability and mRNA expression patterns of developmentally important genes in bovine reconstructed embryos using a mouse fibroblast cell nucleus. While 20% nuclear transferred embryos with bovine fibroblast developed to morulae/blastocysts, a few(2-5%) nuclear transferred bovine embryos with mouse fibroblast developed to morula. (omitted)

• 한국수정란이식학회지
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• 제19권3호
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• pp.191-199
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• 2004

본 연구는 돼지 태아 섬유아세포유래 공여세포를 미세주입에 의해 주입 후 재 조합한 핵 이식 배에 대한 배양액, 세포주기의 동기화, 배양시간 및 난자의 활성화에 따른 융합율과 체외발생율에 대해 조사하였다. 핵 이식 배를 NCSU-23, TL Hepes 및 TZM-3 배양액으로 1시간 및 8시간 배양하였을 때 배반포로의 분할율은 각각 15.6%, 14.0%, 15.0% 및 13.9%, 10.5%, 13.3%로서 배양액 및 시간에 따른 분할율의 유의적인 차이는 없었다. 공여핵원용 세포를 0, 8, 15시간 배양했을 때 G2/M기로의 체외발달율은 12.0%, 18.0%, 48.0%였다(p<0.01). 공여핵원용 세포를 12-24시간 배양했을 때 G2/M기로의 체외발달율은 유의한 증가를 나타내지 않았다. 공여핵원용 세포를 10% FBS + NCSU-23 배양액으로 1-2, 6-8, 12-14일간 배양 후 핵 이식한 배의 융합율은 각각 60.0%, 73.3%, 62.5%였으며, 분할율은 각각 36.0%, 56.7%, 50.0%였다. 0.5% FBS + NCSU-23, 0.5% + TL-Heaps 및 0.5% + TZM-3 배양액으로 5% $O_2$조건 하에서 배양하였을 때 핵 이식배의 $\geq$2 cell 및 배반포로의 발생율은 각각 12.5$\pm$1.6%, 11.1$\pm$1.8%, 11.7$\pm$1.0%였으며, 10% $O_2$조건 하에서 배양하였을 때 핵 이식배의 $\geq$2 cell 및 배반포로의 발생율은 각각 10.5$\pm$1.5%, 9.8$\pm$1.4%, 10.0$\pm$0.8%였다 배양액과 $O_2$ 조건에 따른 유의한 발생율에 차이는 인정되지 않았다.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2003년도 제3회 발생공학 국제심포지움 및 학술대회
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• pp.136-136
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• 2003

• Asian-Australasian Journal of Animal Sciences
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• 제22권2호
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• pp.194-200
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• 2009

The aim of this study was to investigate the effects of donor cell passage, size and type on the development of nuclear transfer embryos. Porcine cumulus cells, fetal fibroblasts and oviductal epithelial cells from 1-2, 3-6 and 7-10 passages were used for the nuclear transfer. In the oocytes with the cumulus donor cells, fusion and cleavage rates of oocytes and cell numbers per blastocyst among the three different passage groups did not show any differences, but the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher than those from 7-10 passage group. The rates of fusion, cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the embryos with the sizes of <20 and 20 ${\mu}m$ cumulus donor cells compared to the >20 ${\mu}m$ cumulus donor cell. In the oocytes with the fetal fibroblast donor cells, the rate of blastocyst formation from the 3-6 passage group was higher than from 1-2 and 7-10 passage groups. The embryos with the size of 20 $\mu{m}$ fetal fibroblast donor cell showed higher rate of blastocyst formation compared to those with <20 and >20 ${\mu}m$ donor cells. In the oocytes with the oviductal epithelial cells, the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher compared to those from 7-10 passage group. The embryos with the sizes of <20 and 20 ${\mu}m$ oviductal epithelial donor cells had a higher rate of blastocyst formation compared to those with >20 ${\mu}m$ donor cell. Fusion and cleavage rates of oocytes, and cell numbers per blastocyst among the three different donor cell types from the 3-6 passage did not show any differences. However, the rate of blastocyst formation of somatic cell nuclear transfer (SCNT) embryos with the fetal fibroblast donor cell was higher than that of blastocyst formation of SCNT embryos with the cumulus and oviductal epithelial donor cells.

• Archives of Plastic Surgery
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• 제32권3호
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• pp.363-368
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• 2005

It was assumed that the effect of estrogen on wound healing would be variable according to patient's gender and age since estrogen is a sex steroid. This study was designed to determine the variability of the effect of estrogen on proliferation of human dermal fibroblasts and collagen synthesis which are most important in wound healing considering patient's gender and age. Fibroblasts were isolated from the dermis of female patients in premenstrual, menstrual, or postmenopausal age group and that of male patients. The isolated fibroblasts were cultivated in the presence of estrogen($1.0{\mu}g/ml$). The cells were seeded at $5.0{\times}10^3cell/well$ in Dulbecco's Modified Eagle's Medium/Ham's F-12 nutrient including 5% fetal bovine serum in 96-well plates. The cells were incubated for 3 days. For fibroblast proliferation MTT assay method was used. To measure the production of collagen, the collagen type I carboxy- terminal propeptide enzyme immunoassay was carried out. Estrogen stimulated the proliferation of fibroblasts in female patients, but not in male patients. The greatest cell proliferation and collagen synthesis was seen at women in menstrual and postmenopausal age. These results demonstrated that effects of estrogen on dermal fibroblast proliferation and collagen synthesis were variable with gender and age.

• 한국가축번식학회지
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• 제24권1호
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• pp.49-57
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• 2000

본 연구는 소 태아섬유아세포를 이용하여 핵이식 후 세포의 휴면처리, 세포의 passage 수 및 세포의 기원이 복제란의 발육에 미치는 영향을 검토하였다.3.57개 월령 한우 수컷 태아의 피부 및 간 조직에서 세포를 채취하여 체외에서 4∼6 회 계대배양 후 동결하였다가 핵이식에 사용하였다. 세포는 핵이식 전에 혈청기아처리를 하였으며, 대조구로는 활발히 분열 중의 무처리 세포를 사용하였다. Donor 세포는 미수정란의 탈핵세포질에 이식 후 전기융합 및 활성화를 실시하였고, 재구축배는 7∼9 일간 체외배양하여 발육농을 검토하였다. 배반포로 발육된 일부 복제란은 발정 7∼8 일째의 수란우에 이식하였다. 복제란의 배반포 발육율은 혈청기아 처리구 (25.3%)가 무처리구 (15.9%)에 비하여 유의적으로 높았으나 (P＜0.05), 세포의 passage 수 (4∼6회) 에 관계없이 23.1∼25.0%로 나타났고, 피부 및 간유래 복제란의 배반포 발육율도 23.8∼25.2% 로 두 조직세포 간에 차이가 없었다. 복제란의 이식 후 60일 및 120일에 수란우의 34.4% 및 15.6%가 각각 임신이 확인되었으며, 초자화동결된 태아 피부세포 복제란으로부터 1두의 수컷 송아지가 생산되었다. 본 연구의 결과는 복제란의 체외발육능이 세포의 휴면처리에 의하여 향상되었으나, 세포의 passage 수 (4∼6 회) 및 세포의 두 기원 (피부 및 간) 에 의해서는 영향을 받지 않으며, 태아 피부세포 유래 복제란으로부터 산자가 생산될 수 있음을 확증한다.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.107-111
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• 2009

The transfection efficiency of a transgene into pig and goat fetal fibroblast cells (PFF and GFF, respectively) was tested using co-transfection of a human tissue-type plasminogen activator (tPA) transgene and neomycin-resistant ($Neo^r$) gene, followed by G418 selection. To initially test G418 resistance, GFF and PFF were incubated in culture medium containing different concentration of G418 for 2 weeks, and cell survival was monitored over time. Based on the obtained results, the concentrations chosen for G418 selection were 800 ug/ml and 200 ug/ml for GFF and PFF, respectively. For co-transfection experiments, the pBC1/tPA and $Neo^r$ vectors were co-transfected into GFF and PFF ($1{\times}10^6$ cells in each case) using the FuGENE6 transfection reagent, and resistant colonies were obtained following 14 days of G418 selection. We obtained 96 and 93 drug-resistant colonies of GFF and PFF, respectively, only 54 and 39 of which, respectively, continued proliferating after drug selection. PCR-based screening revealed that 23 out of 54 analyzed GFF colonies and 5 out of 39 analyzed PFF colonies contained insertion of the tPA gene. Thus, the experimentally determined transfection efficiencies for tPA gene co-transfection with the $Neo^r$ gene were 42.6% for GFF and 12.8% for PFF. These findings suggest that co-transfection of a transgene with the $Neo^r$ gene can aid in the successful integration of the transgene into fetal fibroblast cells.