• 한국수정란이식학회지
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• 제27권1호
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• pp.9-14
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• 2012

This study was conducted to analyze the transgenic efficiency and sex ratio in ${\alpha}$-1,3-galactosyltransferase (GalT) knock-out (KO) transgenic pigs according to generation. GalT KO piglets were produced by artificial insemination or natural mating. The transgenic confirmation of GalT KO was evaluated by PCR amplification using specific primers. After electrophoresis, three types of bands were detected such as 2.3 kb single band (Wild), 2.3 and 3.6kb double bands (GalT KO -/+; heterozygote), and 3.6kb single band (GalT KO -/-; homozygote). Transgenic efficiency in F1 generation was 64.5% (23/35) of GalT KO (-/+). In F2 generation, GalT KO transgenic efficiency was 36.4% (21/57, Wild), 47.5% (28/57, GalT KO -/+), and 16.1% (8/57, GalT KO -/-), respectively. Interestingly, no homozygote piglets were born in 6 deliveries among total 11 deliveries, although they were pregnant between male (M) and female (F) $F_1$ heterozygote. In the 5 litters including at least one GalT KO -/- piglet, the transgenic efficiency was 13.3% (2/24, Wild), 51.3% (14/24, GalT KO -/+), and 35.3% (8/24, GalT KO -/-), respectively. The sex ratio of M and F was 40:60 in $F_1$ and 49:51 in $F_2$ generation, respectively. Based on these results, GalT KO transgenic pigs have had a reproductive ability with a normal range of transgenic efficiency and sex ratio.

• 한국수정란이식학회지
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• 제27권4호
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• pp.205-209
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• 2012

Transgenic rats and mice are useful experimental animal models for medical research including human disease model studies. Somatic cell nuclear transfer (SCNT) technology is successfully applied in most mammalian species including cattle, sheep, pig and mouse. SCNT is also considered to increase the efficacy of transgenic/knockout mouse and rat production. However, in the area of reproductive biotechnology, the rodent model is inadequate because of technical obstacles in manipulating the oocytes including intracytoplasmic sperm injection and SCNT. In particular, success of rat SCNT is very limited so far. In this review, the history of rodent cloning is described.

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

• Reproductive and Developmental Biology
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• 제37권2호
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• pp.85-89
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• 2013

For more than two decades, the intracytoplasmic sperm injection (ICSI) technique has been used as a valuable tool to provide opportunities for studying fertilization, treating human infertility, and producing transgenic animals. Not only in facilitating fertilization but also in propagating mammalian species, ICSI has enhanced the potential of assisted reproductive technologies in human. Polyspermic fertilization has been one of major problems in pig reproduction, but the ICSI helped to solve the problem, and used widely to generate transgenic piglets. Although the ICSI technique is considered to be a very useful tool in assisted reproductive technologies, including generation of transgenic animals, there are some disadvantages using the technique. In this review, we describe the ICSI technique and its application in animal production and human infertility, and discuss advantage and disadvantage of the technique in mammals.

• 한국동물생명공학회지
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• 제35권1호
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• pp.42-49
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• 2020

As a preclinical study, many researchers have been attempted to convert the porcine PSCs into several differentiated cells with transplantation of the differentiated cells into the pigs. Here, we attempted to derive neuronal progenitor cells from pig embryonic germ cells (EGCs). As a result, neuronal progenitor cells could be derived directly from pig embryonic germ cells through the serum-free floating culture of EB-like aggregates (SFEB) method. Treating retinoic acid was more efficient for inducing neuronal lineages from EGCs rather than inhibiting SMAD signaling. The differentiated cells expressed neuronal markers such as PAX6, NESTIN, and SOX1 as determined by qRT-PCR and immunostaining. These data indicated that pig EGCs could provide valid models for human therapy. Finally, it is suggested that developing transgenic pig for disease models as well as differentiation methods will provide basic preclinical data for human regenerative medicine and lead to the success of stem cell therapy.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.60-60
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• 2002

본 연구는 사람의 조혈촉진유전자가 형질전환된 돼지의 유즙내 hEPO 발현농도를 확인하고 3종의 동물 (mouse, rat, swine)에서 hEPO 의 생리활성을 분석하기 위하여 실시하였다. 새롬이 Fl 암컷의 유즙을 원심분리하여 지방을 제거한 후 hEPO 의 농도를 측정하고, PBS 로 유즙내 hEPO 농도가 100 IU/㎖가 되도록 희석하였다. Mouse (ICR)는 피하주사로 20 IU씩 2일 간격으로 3회 (0, 2, 4 일차 ) 주사하였으며, 주사 후 4회에 걸쳐 (0, 2, 4, 7 일차) 혈액을 채취하여. 분석하였다. (중략)

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.88-88
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• 2002

Erythropoietin (EPO)는 조혈작용 (erythropoiesis)을 나타내는 호르몬으로서 사람의 빈혈치료제로 사용되며, 포유통물 중 사람, 생쥐 등의 유즙 내에 혈청 EPO 와 동일한 크기로 다량으로 존재한다고 보고된 바 있다. 생쥐의 WAP promoter를 이용하여 사람의 조혈촉진제인 EPO를 유즙으로 생산하는 형질전환돼지 (새롬이)의 유즙을 분석하기 위해 SDS-PAGE와 Western blotting 을 수행하였다. 먼저, 형질전환돼지의 유즙으로부터 원심분리에 의해 지방층을 제거한 후, 16.5% polyacrylamide gel 에서 PAGE를 수행하였다. (중략)

• 한국가축번식학회지
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• 제27권3호
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• pp.215-223
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• 2003

본 연구는 체외성숙된 돼지난포란을 액상정액으로 수정시 수정시간과 배양배지가 난포란의 발달에 미치는 영향을 조사하기 위하여 실시하였다. 정자농후정액 (30∼60 ml)을 채취하여 실온에서 2시간 정도 서서히 냉각시킨 후, 정액을 15 ml 튜브에 담아 800${\times}$g로 10분간 원심분리하였다. 상층액은 버리고 하부의 정자는 5 ml LEN 희석액으로 1${\times}$$10^{9}$ 전자/ml가 되도록 재희석하였다. 희석된 정액은 4$^{\circ}C$ 냉장고에 보존하였다. 미성숙 난모세포의 성숙에 사용된 배지는 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin B$_{12}$ , 25 mM HEPES, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml PMSG, 10 IU/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate그리고 10% pFF를 첨가한 TCM-199 배지였다. 22시간 성숙 배양한 후 난모세포는 cysteamine과 hormone들을 배제한 후 38.5$^{\circ}C$, 5% $CO_2$ incubator에서 22시간 더 성숙시켰다. 성숙된 난모세포는 채취 후 2일간 4$^{\circ}C$에 보존된 액상정액으로 수정되었다. 난모세포는 500 $\mu$l mTBM 수정 배지에서 1${\times}$$10^{6}$ 정자/ml의 농도로 1, 3, 6 그리고 9시간 동안 수정시켰다. 그 후 난모세포는 500 $\mu$l NCSU-23, Hopes buffered NCSU-23, PZM-3 그리고 PZM-4 배양배지에 옮겨서 6, 48 그리고 144시간을 더 배양하였다. 정자침투율, 웅성전핵형성율 그리고 난모세포의 난할율은 6 및 9시간 수정시간에서 1 및 3시간 수정시간 보다 높았다. 6시간 수정시 배반포형성율 (33.6%)은 1, 3 그리고 9시간 수정시 배반포형성율 (11.4, 23.0 그리고 29.6%) 보다 높았다. 배반포의 평균세포수는 6, 9, 3 그리고 1시간 수정시 각각 32.9, 27.6, 26.3 그리고 24.4개 였다. 분할된 난모세포의 배반포형성율 그리고 배반포의 평균세포수는 NCSU-23, PZM-3 그리고 PZM-4 배양배지보다 HEPES buffered NCSU-23 배양배지가 우수하였다. 결론적으로 4$^{\circ}C$ 보존 돼지액상정액은 체외성숙된 돼지 난모세포의 체외수정에 사용될 수 있음이 입증되었다. 또한 체외성숙된 돼지 난모세포는 500 $\mu$l mTBM 수정배지에서 1${\times}$$10^{6}$ 정자/ml로 6시간 공배양시키는 것이 바람직하며, HEPES buffered NCSU-23 배양배지에서 배양하는 것이 좋다는 결과를 얻었다.

• Asian-Australasian Journal of Animal Sciences
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• 제21권8호
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• pp.1127-1133
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• 2008

This study was designed to develop a method of liquid storage of boar sperm at $4^{\circ}C$ by using the modified Beltsville F5 (BF5) diluent with bovine serum albumin (BSA) and N-acetyl-D-glucosamine. Boar sperm were stored in lactose-egg yolk and N-acetyl-D-glucosamine (LEN), BF5 and Golden-Pig liquid 4 (GPL4) diluents at $4^{\circ}C$ for 5 days and were examined for sperm viability, adenosine triphosphate (ATP) and in vitro fertilization (IVF). The percentage of sperm viability in GPL4 diluent was higher than in LEN and BF5 diluent from 1 to 5 days of storage at $4^{\circ}C$. The percentage of sperm viability steadily declined from 1 to 5 days of storage in the three different diluents. Sperm ATP in GPL4 diluent was higher than in LEN and BF5 diluents from 1 to 5 days of storage. Sperm ATP rapidly declined after 5 days of storage in the three different diluents. Porcine oocytes matured in vitro were inseminated with different sperm concentrations of liquid semen stored for 3 days in GPL4 diluent. The percentage of monospermic oocytes did not show any differences from 2.5 to $20{\times}10^5$ sperm/ml. However, the percentage of polyspermic oocytes in the sperm concentration of $2.5{\times}10^5$ sperm/ml was lower than in concentrations of 5, 10 and $20{\times}10^5$5 sperm/ml. The percentage of blastocysts from the cleaved oocytes at $2.5{\times}10^5/ml$ sperm concentration was significantly lower than at 5, 10 and $20{\times}10^5sperm/ml$ concentrations. In conclusion, GPL4 diluent can be stored at $4^{\circ}C$ for 5 days and showed higher sperm viability and sperm ATP concentration compared with LEN and BF5 diluents. Also, we found that GPL4 diluent can be used for IVF of porcine oocytes.

• 한국임상수의학회지
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• 제28권1호
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• pp.57-62
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• 2011

The shortage of transplantable kidneys has many efforts to regenerate bioartificial kidneys using transgenic animals and diverse kinds of scaffolds which are important tools for cell seeding. However, there are many limitations for clinical applications so far. Recently, decellularized bioscaffolds using animal organs come into spotlight because of its many superior advantages. In current study, we produced decellularized kidney bioscaffolds of pig which is an attractive animal as a clinical model for human. We decellularized pig kidneys with 1% SDS detergent solution using peristaltic pump systems for 12h. After decellularization process, the kidney bioscaffolds preserved intact 3D morphology including glomerular structure and almost DNA from pig was entirely removed. In addition, this process could preserve micro vascular network which is necessary for cell survival. Although, additional studies for recellularization and transplantation should be required, the decellular vascularized kidney bioscaffolds might have many potentials for kidney regeneration.