• 한국임상수의학회:학술대회논문집
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• 한국임상수의학회 2008년도 춘계학술대회
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• pp.91-91
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• 2008

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.63-63
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• 2003

This study was carried out to investigate the effects of liquid boar sperm stored at 4$^{\circ}C$ on sperm motility, normal acrosome, and in-vitro fertilization and culture of pig oocytes matured in-vitro. The sperm-rich fraction (30~60 ml) of ejaculate was collected into an insulated vacuum bottle. Semen was slowly cooled to room temperature (20~23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min at 800$\times$g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of lactose, egg yolk and N-acetyl-D-glucosamine (LEN) diluent to provide 1.0$\times$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$ and preserved for 5 days to examine sperm motility and normal acrosome. The medium used for oocyte maturation was modified tissue culture medium (TCM) 199. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 6 h in 500 ${mu}ell$ mTBM fertilization media with 0.2, 1, 5 and 10$\times$10$^{6}$ /ml sperm concentration, respectively. At 6 h after IVF, oocytes were transferred into 500 ${mu}ell$ Hepes-buffered NCSU-23 culture medium for further culture of 6, 48 and 144 h. There were significant differences in sperm motility and normal acrosome among preservation days and incubation times, respectively. The rates of sperm penetration and polyspermy were higher in 5 and 10$\times$10$^{6}$ sperm/ml than in 0.2 and 1$\times$10$^{6}$ sperm/ml. Male pronuclear formation was lower in 0.2$\times$10$^{6}$ sperm/ml than in 1, 5 and 10$\times$10$^{6}$ sperm/ml. Mean numbers of sperm in penetrated oocyte were highest in 10$\times$10$^{6}$ sperm/ml compared with other sperm concentrations. The rate of blastocysts from the cleaved oocytes (2~4 cell stage) was highest in 1$\times$10$^{6}$ sperm/ml compared with other sperm concentrations. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend 1$\times$10$^{6}$ ml sperm concentration for in-vitro fertilization of pig oocytes.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.9-19
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• 2003

Pig organ is thought to be the most suitable nonhuman organ for xenotransplanstation. However, one of the major constraints to using pig organs for xenotransplantation is human natural antibody-mediated hyperacute rejection (HAR). Elimination of a(1,3) galactosyltransferase (GGTA1) from the pig is expected to be a solution to the problem of hyperacute rejection. Many efforts have made characterization of GGTA1 in structure and function, improvement in the technique of DNA transfection of somatic cells and advancement of the pig NT, a specific modification has been made to one copy of the GGTAl gene by Missouri group in 2002 To date because homozygousity of the genetic modification has been achieved in this gene, the role of gala(1,3) gal specific natural antibody in HAR and the efficacy of xenotransplantation in a nonhuman primate model will be addressed. Of other genes are found to be involved in rejection of pig donors by primates, the technology will be available to modify those genes so that rejection can be overcome.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2003년도 제3회 발생공학 국제심포지움 및 학술대회
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• pp.9-19
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• 2003

Pig organ is thought to be the most suitable nonhuman organ for xenotransplanstation. However, one of the major constraints to using pig organs for xenotransplantation is human natural antibody-mediated hyperacute rejection (HAR). Elimination of a(1,3) galactosyltransferase (GGTA1) from the pig is expected to be a solution to the problem of hyperacute rejection. ry1any efforts have made characterization of GGTA1 in structure and function. improvement in the technique of DNA transfection of somatic cells and advancement of the pig NT, a specific modification has been made to one copy of the GGTA1 gene by Missouri group in 2002. To date because homozygousity of the genetic modification has been achieved in this gene, the role of gala(1,3) gal specific natural antibody in HAR and the efficacy of xenotransplantation in a nonhuman primate model will be addressed. If other genes are found to be involved in rejection of pig donors by primates, the technology will be available to modify those genes so that rejection can be overcome.

• 한국수정란이식학회지
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• 제30권4호
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• pp.345-350
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• 2015

In the last 10 years, porcine somatic cell nuclear transfer to generate transgenic pig has been performed tremendous development with introduction and knockout of many genes. However, efficiency of porcine somatic cell nuclear transfer is still low and embryo transfer (ET) is one of important step for production efficiency. In porcine ET for production of transgenic cloned pig, we can consider many of points to increase production rates. In respect of seasonality and weather, porcine ET usually is not performed in summer and winter. Cloned transgenic embryos must be transferred into reproductive tracts of recipients where embryos are located after natural fertilization with similar estrous cycle. If cloned embryos with 2~4 cell stage are transferred, they must be transferred into oviducts in periovulatory stage. Number and deposition sites of transferred cloned embryos are important. And we must compare the methods of ET between surgical and non-surgical ones in respect of production efficiency. Sow recipients after natural estrus is most preferred recipients however its cost is must be considered. Here we will review many of current studies about porcine embryo transfer to increase production efficiency of transgenic pigs and strategies for further studies.

• 한국가축번식학회지
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• 제18권3호
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• pp.191-197
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• 1994

광수용기에서 특이적 발현을 유도하는 로돕신 유전자에 존재하는 다양한 점 돌연변이 의해서 일반적으로 색소성 망막염을 초래한다. 이 질병의 측징은 광수용기 세포의 퇴화로 인한 광수용기의 기능상실을 초래하고, 결과적으로 시각상실을 유발한다. 로돕신 유전자는 광수용기의 바깥분절에 존재하며, 빛 에너지를 흡수하여, 시각반응을 야기한다. 본 실험에 사용된 로돕신 유전자는 총 12.5kb의 돼지 로돕신 게놈 유전자로서, 망막에서 특이발현을 유도하는 4kb의 프로모터, 돌연변이를 지닌 5.6kb의 유전자, 그리고 poly A site로 구성되어 있다. 호르몬 투여에 의해 과배란이 유기된 C57BL/6J 생쥐 난자의 웅성전핵에 미세주입법에 의해 도입한 후, 위임신한 생쥐의 난관에 이식하였다. 그 결과 태어난 산자로부터 돌연변이 로돕신 유전자를 지닌 6마리의 형질전환동물을 PCR과 Southern blot analysis를 통하여 확인할 수 있었다. 또한, 이 형질전환 동물들은 로돕신 유전자를 그 자손에게 안정적으로 전달하는 것을 확인하였다. 본 연구는 광수용기 퇴화의 원인규명 및 치료방법을 연구하기 위하여 돌연변이를 지닌 질환모델동물을 생산하는데 있다. 이들 형질전환 동물은 로돕신 유전자의 돌연변이에 의해 야기되는 시각상실의 기전에 관한 연구와 사람의 장님을 치료하기 위한 질환모델동물로서 유용하게 이용될 것이다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.216-216
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• 2004

Genetically modified domestic animals have many potential applications ranging from basic research to production agriculture. One of the goals in transgenic animal production schemes is to reliably predict the expression pattern of the foreign gene. Establishing a method to screen genetically modified embryos for transgene expression before transfer to surrogates may improve the likelihood of producing offspring with the desired expressing pattern. (omitted)

• 한국초지학회:학술대회논문집
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• 한국초지조사료학회 2005년도 학술심포지엄, 제43회 학술발표회
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• pp.152-153
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• 2005

• 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.

• 한국발생생물학회지:발생과생식
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• 제21권2호
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• pp.157-165
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• 2017

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-human-primate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were $127{\pm}18.9$. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as ${\alpha}-1,3-galactosyltransferase$ knock-out /hCD46 for xenotransplantation.