• Title/Summary/Keyword: transgenic pig

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Production of ${\alpha}$1,3-Galactosyltransferase (GalT) Double Knock-out (-/-) Transgenic Pigs for Xenotransplantation (${\alpha}$1,3-Galactosyltransferase(GalT) 유전자가 완전 Knock-out(-/-)된 바이오장기용 형질 전환 돼지 생산)

  • Hwang, Seong-Soo;Oh, Keun-Bong;Kim, Dong-Hoon;Woo, Jea-Seok;Shim, Ho-Sup;Yun, Ik-Jin;Park, Jin-Ki;Im, Gi-Sun
    • Journal of Embryo Transfer
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    • v.27 no.1
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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.

Investigation of Deletion Variation and Methylation Patterns in the 5' LTR of Porcine Endogenous Retroviruses

  • Jung, K.C.;Simond, D.M.;Moran, C.;Hawthorne, W.J.;Jeon, J.T.;Jin, D.I.;Lee, J.H.
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.11
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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.

Application of Intracytoplasmic Sperm Injection in Mammals

  • Li, Xiao Xia;Lee, Kyung Bon;Lee, Ji Hye;Kim, Keun Jung;Park, Kang Sun;Kim, Min Kyu
    • Reproductive and Developmental Biology
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    • v.37 no.2
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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.

Somatic Cell Nuclear Transfer in Rodents, the Little Big Animals

  • Roh, Sangho
    • Journal of Embryo Transfer
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    • v.27 no.4
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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.

Generation of Neural Progenitor Cells from Pig Embryonic Germ Cells

  • Choi, Kwang-Hwan;Lee, Dong-Kyung;Oh, Jong-Nam;Kim, Seung-Hun;Lee, Mingyun;Jeong, Jinsol;Choe, Gyung Cheol;Lee, Chang-Kyu
    • Journal of Animal Reproduction and Biotechnology
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    • v.35 no.1
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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.

사람의 조혈촉진유전자(hEPO)가 도입된 형질전환돼지의 유즙내 hEPO의 생리활성분석

  • 박진기;이연근;전익수;최영진;김정호;정길생;이훈택;민관식;장원경
    • Proceedings of the KSAR Conference
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    • 2002.06a
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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)

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Monoclone 항체를 이용한 사람 EPO 형질전환돼지의 유즙내 발현단백질 분석

  • 이연근;정희경;이현기;이풍연;박진기;민관식;김진회;장원경
    • Proceedings of the KSAR Conference
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    • 2002.06a
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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)

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Effects of Fertilization Time and Culture Medium of Pig Oocytes Matured In Vitro by liquid Boar Sperm Stored at $4^{\circ}C$ (체외성숙된 돼지난포란을 $4^{\circ}C$ 보존 액상정액으로 체외수정시 수정시간과 배양배지의 영향)

  • Park, C. S.;Y. J. Yi;Kim, M. Y.;Y. J. Chang;Lee, S. H.;D. I. Jin
    • Korean Journal of Animal Reproduction
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    • v.27 no.3
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    • pp.215-223
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    • 2003
  • This study was to investigate the effects of fertilization time and culture medium of pig oocytes matured in-vitro by liquid boar sperm. The sperm rich fraction (30∼60 ml) 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 800 ${\times}$ g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of the LEN diluent to provide 1.0${\times}$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$. The medium used for oocyte maturation was TCM-199 supplemented with 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 hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. 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 1, 3, 6 and 9 h in 500 ${mu}ell$ mTBM fertilization media with 1.0${\times}$10$^{6}$ sperm/ml concentration, respectively. Thereafter, oocytes were transferred into 500 ${mu}ell$ NCSU-23, HEPES buffered NCSU-23, PZM-3 and PZM-4 culture media, respectively, for further culture of 6, 48 and 144 h. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times for 6 and 9 h than in those for 1 and 3 h. The rates of cleaved oocytes were higher in the fertilization times for 6 and 9 h (85.0 and 84.6%) than in those for 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time for 6 h (33.6%) than in that for 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9, 27.6, 26.3 and 24.4 in the fertilization times for 6, 9, 3 and 1 h, respectively. The rate of blastocyst from the cleaved oocytes and the number of cells per blastocyst were higher in HEPES buffered NCSU-23 culture medium than in NCSU-23, PZM-3 and PZM-4 culture media. 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 the coincubation time of 6 h in 500 ${mu}ell$ TBM fertilization medium with 1${\times}$10$^{6}$ sperm/ml concentration and the HEPES buffered NCSU-23 culture medium for in-vitro fertilization of pig oocytes matured in-vitro.

Comparison of Viability, ATP and In vitro Fertilization of Boar Sperm Stored at 4℃ in the Three Different Diluents

  • Yi, Y.J.;Li, Z.H.;Kim, E.S.;Song, E.S.;Cong, P.Q.;Zhang, Y.H.;Lee, S.H.;Lee, J.W.;Park, Chang-Sik
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.8
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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.

Decellularized Bioscaffold of Pig Organs: A Tool for Patient-specific Organogenesis Using Induced Pluripotent Stem Cells (탈세포화 기법을 이용한 돼지 바이오 스캐폴드: 환자 맞춤형 장기재생을 위한 역분화 줄기세포 동물모델)

  • Park, Kyung-Mee;Kwak, Ho-Hyun;Nam, Hyun-Suk;Park, In-Chul;Jeon, Yong-Hwan;Park, Sung-Min;Lee, Seung-Tae;Woo, Jae-Seok;Woo, Heung-Myong
    • Journal of Veterinary Clinics
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    • v.28 no.1
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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.