• 한국가축번식학회지
• /
• 제27권4호
• /
• pp.275-280
• /
• 2003

This study was carried out to investigate the effect of cysteamine addition during in vitro maturation, fertilization and culture of porcine oocytes. Oocytes were matured for the first 22 h in mTCM -199 media supplemented with or without 150 $\mu$M cysteamine. They then were matured for an additional 22 h in mTCM-199 media without hormones supplemented with or without 150 $\mu$M cysteamine. When cumulus-oocyte complexes (COCs) were matured in the mTCM-199 media supplemented with cysteamine, the rates of GVBD and maturation (metaphase II) were enhanced as compared to the media without the addition of cysteamine. Also, when COCs were matured in the mTCM-199 media supplemented with cysteamine, the rates of sperm penetration, male pronucleus formation, cleavage and blastocyst formation after in vitro fertilization were enhanced as compared to the media without the addition of cysteamine. In conclusion, it was suggested that oocytes matured for the first 22 h in mTCM-199 media supplemented with 150 $\mu$M cysteamine increased the rates of metaphase II, sperm penetration, male pronucleus and blastocyst formation were higher as compared to the media without addition of cysteamine.

• 한국가축번식학회지
• /
• 제27권2호
• /
• pp.179-185
• /
• 2003

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.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2001년도 춘계학술발표대회
• /
• pp.59-59
• /
• 2001

Interleukin-10 (IL-10) is known as a regulator of inflammation and pathogenesis in mammalian organs, but its precise role is little known in the mammary gland. Our initial experiment showed that IL-10 expression levels in mice decreased at the lactation stage otherwise increased at the involution stage. To reveal the effects of IL-10 on the involution of mammary gland, expression profiles of the apoptosis-related genes were examined in transgenic mice expressing human IL-10 as well as in knock-out mice (IL-10-/-). Mild inflammatory legions by lymphocytes were observed in the mammary glands of transgenic lines at the lactation stage. The expression of TRAIL (Tumor necrosis factor-Related Apoptosis-Inducing Ligand) among the apoptosis-related genes was highly elevated in the transgenic mice while others were not significantly changed. Furthermore, TRAIL was down regulated by four fold in the IL-10-/- mice at the involution stage. The expression of DR4 was elevated at the involution stage of normal mice. DR4 was detected in the milk of transgenic mice but absent in that of normal mice. Our results proposed that the elevated IL-10 at the involution stage recruit lymphocytes and induce TRAIL and DR4 genes, therefore, lead to enter involution stage of mammary glands.

• Asian-Australasian Journal of Animal Sciences
• /
• 제21권11호
• /
• pp.1572-1575
• /
• 2008

The xenotransplantation of pig organs and cells can be related with a risk of transmission of infectious diseases to human. Previous findings indicate that the regulatory region of PERV for retroviral transcription, replication and integration into the cellular DNA is located on the 5' Long Terminal Repeat (LTR). The objective of this study is the investigation of methylation and deletion status of the PERV 5' LTR region which can be used for regulating PERV expression. We compared the sequences of genomic DNA and bisulfite-treated genomic DNA from PK-15 cells expressing PERV to observe the methylation status of the 5' LTR. Our results showed that the CpG sites of U3 were methylated and methylation was inconsistent in the R and U5 regions. Also, variable numbers of 18 bp repeats and 21 bp repeats were detected on 5' LTR by sequencing analysis. The consistent U3 methylation might be indicative of host suppression of expression of the retroviruses.

• 한국수정란이식학회지
• /
• 제30권3호
• /
• pp.225-228
• /
• 2015

Understanding the behavior of transgenes introduced into oocyte or embryos is essential for evaluating the methodologies for transgenic animal production. To date, many studies have reported the production of transgenic pig embryos with, however, low efficiency in environment of blastocyst production. The aim of present study was to determine the expression and duration of transgene transferred by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of GFP and then for the transmission of the transgene. Briefly, fresh spermatozoa were bound to exogenous DNA after treatment by Triton X-100 and Lipofectin. When ICSI-MGT was performed using sperm heads with tails removed, the yield of blastocyst (25.3%), treated with Lipofectin (18.8%) and Triton X-100 (19.2%) were observed. Treatments of Lipofectin or Triton X-100 did not further improve the rates of blastocysts. Moreover, the apoptosis rates of embryos were obtained from the control and LIpofectin groups (8.7%, 9.7%, respectively), but were significantly higher in the Triton X-100 group (13.0%). Our results demonstrated that ICSI-MGT caused minimal damage to oocytes that could develop to full term. Moreover, the embryos derived by ICSI-MGT have shown prolonged exogenous DNA expression during preimplantation stage in vivo. However, more efforts will be required to improve the procedures of both sperm treatments cause of high frequency of mosaicisms.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
• /
• pp.94-94
• /
• 2002

Increasing competition in the livestock industry has forced producers to cut costs by adopting new technologies aimed out increasing production efficiency. Non-ruminant livestock do not express fibrolytic enzymes. The major plant cell wall components of cereals, primarily β-glucans and arabinoxylans, form gel-like structures in the small intestines that trap nutrients. The viscous polysaccharides can also cause severe gastrointestinal disorders. (omitted)

• Reproductive and Developmental Biology
• /
• 제32권1호
• /
• pp.39-43
• /
• 2008

Most studies on transgenic bioreactors have focused on expression levels of interest genes. In this study we examined whether transgenic bioreactors would inherit expression level of the Oansgene to long-term generations independently of transgene sources. We employed three transgenic mice, which were separately reported, carrying different transgenes and copy numbers, 27 kb of hLF and 22 kb of hIL-10 genomic sequences, and 1.3 kb of hTPO cDNA, respectively. Three females of the transgenic lineages crossbred with a wild-type male up to 20 generations to test transgenic frequencies of their progenies and to determine expression levels of the transgenes. Ultimately, transmission rates of kLF, hIL-10, and hTPO were $64.3{\pm}7.0$, $59.3{\pm}9.8$, and $56.1{\pm}9.7$, respectively, appeared following Mendelian pattern of inheritance. Notably, we found that levels of expressions of hLF, hIL-10, and hTPO in milk were sustained to high numbers of generations. No transgene silencing of expression was observed in every generations of all transgenic mice. In conclusion, we suggest that once established animal bioreactors could consistently transmit the transgene to continual generations, without loss of expressional activity, independently of transgene sources.

• Reproductive and Developmental Biology
• /
• 제35권3호
• /
• pp.239-245
• /
• 2011

The overexpression of Phosphoprotein Enriched in Astrocytes (PEA15) gene is commonly found in human diabetic patients. The overexpression of this gene in skeletal muscle and fat tissues have been reported to cause insulin resistance, thereby impairing insulin stimulated glucose uptake. We introduced a gene of mouse PEA15 (mPEA15) and enhanced green fluorescent protein (EGFP) into fertilized one cell pig zygotes using microinjection, and produced a piglet that showed overexpression of mPEA15 in the muscle tissues and expression of EGFP in the ear tissues and hooves. RT-PCR RFLP, southern blot and FISH analysis showed that the tissues carried the transgene. Real-time RT-PCR and western blots demonstrated that PEA15 gene was overexpressed in the various tissues and muscle tissues, respectively. These fads suggest that expression vector system is normally expressed in the transgenic (TG) pigs. To use as animal diseases model for type 2 diabetes, further study is necessary to confirm whether diabetes occur in these TG pigs, especially insulin resistance.

• 한국수정란이식학회지
• /
• 제30권4호
• /
• pp.305-313
• /
• 2015

Xenotransplantation of pig islet regarded as a good alternative to allotransplantation. However, cellular death mediated by hypoxia-reoxygenation injury after transplantation disturb success of this technique. In the present study, we produce transgenic pig expressing human heme oxygenase 1 (HO1) genes to overcome cellular death for improving efficiency of islet xenotransplantation. Particularly, Korean miniature pig breed, Micro-Pig, was used in the present study. Somatic cell nuclear transfer (SCNT) technique was used to produce the HO1 transgenic pig. Six alive transgenic piglets were produced and all the transgenic pigs were founded to have transgene in their genomic DNA and the gene was expressed in all tested organs. Also, in vitro cultured fibroblasts derived from the HO1 transgenic pig showed low reactive oxygen species level, improved cell viability and reduced apoptosis level.

• 한국가금학회지
• /
• 제35권3호
• /
• pp.241-245
• /
• 2008

형질 전환 닭 생산 방법들 가운데 목적 유전자 운반에 탁월한 능력이 있는 것으로 알려진 렌티바이러스는 배반엽 단계 수정란을 이용한 형질 전환 닭 생산 연구에 활발하게 이용되고 있다. 본 연구는 재조합 렌티바이러스를 이용하여 사람의 SOD-3 단백질이 닭의 ovalbumin 프로모터에 의해서 유도되는 형질 전환 닭을 생산하고자 하였다. 사람의 SOD-3 단백질은 호흡 과정에서 체내에서 생성되는 활성산소를 중화시키는 탁월한 기능이 있는 것으로 알려져 있다. 후보 병아리의 생산은 앞서 언급한 유전자를 가지는 $1{\times}10^6$ cfu/mL 재조합 렌티바이러스를 배반엽 단계 수정란의 미세 주입하고 대리난각 배양법을 이용하여 배양기에서 21일 동안 배양하는 방법으로 생산하였다. 유전자를 미세주입한 341개의 수정란에서 78수의 후보 형질 전환 병아리를 생산하였으며, 생산된 후보 형질 전환 병아리들의 유전 분석은 PCR 방법을 이용하여 검증하였다. 유전 분석 결과는 성 성숙에 이른 47수의 수컷들 가운데 2수의 정액에서 사람의 SOD-3 유전자가 존재함을 보였다. 이상의 연구 결과는 완전한 형태의 형질전환 닭 생산의 가능성을 보여주고 있다.