• Reproductive and Developmental Biology
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• 제28권1호
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• pp.71-76
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• 2004

Gene delivery is one of the keen interests in animal industry as well as research on gene functions. Some of the in vivo gene delivery techniques have been successively used in various tissues for the gene therapy and transgenesis. Despite intensive efforts, it still remains to overcome problems of limited local and regional administration and low transgene expression. To improve the efficiency of gene delivery, a new procedure was tested. We injected exogenous DNA containing LacZ into the female or male gonads and then pulsed electric field. Electroporated gonads showed positive X-gal staining in many seminiferous tubules of the porcine fetal gonads. Exogenously introduced LacZ genes were also expressed in female porcine gonad. In addition, we demonstrated efficient gene delivery in gonad of adult mouse. Furthermore, we succeed to generate genetically modified germline cells showing GFP and positive X-gal signals. The results suggest that the newly developed gene delivery is an effective way of in vivo transfection in mammals. The developed gene delivery procedure should be useful in producing transgenic animals when combined with primary cell culture and nuclear transplantation.

• 한국동물생명공학회지
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• 제34권3호
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• pp.232-239
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• 2019

In general, cloned pigs have been produced using the somatic cell nuclear transfer (SCNT) technique with various types of somatic cells; however, the SCNT technique has disadvantages not only in its low efficiency but also in the development of abnormal clones. This study aimed to compare early embryonic development and quality of SCNT embryos with those of induced pluripotent stem cells (iPSCs) NT embryos (iPSC-NTs). Ear fibroblast cells were used as donor cells and iPSCs were generated from these cells by lentiviral transduction with human six factors (Oct4, Sox2, c-Myc, Nanog, Klf4 and Lin28). Blastocyst formation rate in iPSC-NT (23/258, 8.9%) was significantly lower than that in SCNT (46/175, 26.3%; p < 0.05). Total cell number in blastocysts was similar between two groups, but blastocysts in iPSC-NT had a lower number of apoptotic cells than in SCNT (2.0 ± 0.6 vs. 9.8 ± 2.9, p < 0.05). Quantitative PCR data showed that apoptosis-related genes (bax, caspase-3, and caspase-9) were highly expressed in SCNT than iPSC-NT (p < 0.05). Although an early development rate was low in iPSC-NT, the quality of cloned embryos from porcine iPSC was higher than that of embryos from somatic cells. Therefore, porcine iPSCs could be used as a preferable cell source to create a clone or transgenic animals by using the NT technique.

• 대한수의학회지
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• 제50권3호
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• pp.247-251
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• 2010

This study compared the instrument performance and tissue healing of a steel scalpel with a $CO_2$ laser in an animal urinary bladder surgery model. Landrace and Yorkshire mixed breed pigs were used. Two symmetrical incisions were made in urinary bladder of each pig. One incision was made on the left side of ventral aspect on urinary bladder using a steel scalpel, while the other incision was performed on the right side using a $CO_2$ laser with an 8W output power. Each instrument was evaluated clinically for speed, ease of incision, and extent of bleeding. At 7 and 21 days after initial wounding, each wound was taken for histological observations. The scalpel was an easier instrument to use in the confines of the urinary bladder tissue, compared with the laser. However, there was no significant difference between the two groups. The amount of bleeding was less in the laser group but the time of the incisions was shorter with the scalpel. Scalpel incisions showed complete restoration of the epithelium and muscularis. On the other hand, the laser incisions showed incomplete restoration of the epithelium and muscularis. However, most of wound healing in the laser incisions was accomplished according to the time lapse. Although the scalpel produced less damage to the urinary bladder tissue and was easier to handle than the $CO_2$ laser, it did not provide hemostasis that was helpful for use on highly vascular tissue. The $CO_2$ laser provided good hemostasis, but delayed wound healing. In conclusion, the $CO_2$ laser provided better hemostasis and better surgical field than the scalpel. The $CO_2$ laser was used effectively in urinary bladder incision.

• 한국동물위생학회지
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• 제32권1호
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• pp.27-32
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• 2009

We studied the seroprevalence of four respiratory pathogens in Korean swine farms located in Chungnam, Chungbuk, Gyeongnam and Gyeongbuk provinces during the period of spring of 2007 to winter of 2008. Serological tests were performed using commercial ELISA kits. A total of 530 serum samples were tested for the antibodies against porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), Mycoplasma hyopneumoniae (M. hyo) and Actinobacillus pleuropneumoniae (APP). Seroprevalence for four respiratory pathogens were estimated by ELISA-positive rates of the submitted samples. The overall seropositive rates of PRRSV, APP, M. hyo and PCV2 were 32.6%, 10.6%, 38.4% and 88.5%, respectively. By production stage, the seropositive rate for PRRSV was highest in nursery pig populations (46.2%). In contrast, the highest seropositive rates of APP and M. hyo were observed in sow and growing pigs. However, the seroprevalence of PCV2 was ranged from 85.7% to 89.6%, showing no significant difference among the production stages. In the seroprevalence by season, PRRSV, APP and M. hyo infections revealed typical seasonal patterns that the peaks of the seropositive rates were observed between early winter and late spring. In case of PCV2, no particular seasonal patterns were noticed. The pig herds in Gyeongbuk province where PMWS was endemic during the period of survey showed the highest seropositive rates for PRRSV (44.6%), M. hyo (47.5%), and PCV2 (92.7%). Seropositive rates for APP of four provinces were approximately 10%. These results might be valuable for control and prevention of the respiratory diseases and helpful to define strategies related to vaccine applications.

• 한국임상수의학회지
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• 제24권3호
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• pp.300-304
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• 2007

본 연구에서는 돼지에서 tiletamine-zolazepam (TZ) 단독 사용과 azaperone, tiletamine-zolazepam (ATZ) 합제 사용시의 마취효과를 규명하고, 양군에서의 생리학적 parameters를 비교하였다. 두개의 군으로 구분하였으며 건강한 6개의 랜드레이스와 요크셔 교잡종 돼지를 실험에 사용하였다. 교차실험을 하였으며, 각 군 사이의 휴약기간은 2주로 하였다. 1군 (TZ 군): 돼지 6두 ($31.4{\pm}4.83$) kg에 TZ 4.4 mg/kg을 투여하였다. 2군 (ATZ 군): 동일한 돼지 6두 ($43.6{\pm}4.31$ kg)에 azaperone 2 mg/kg을 투여하고, 20분 후에 TZ 4.4 mg/kg을 투여하였다. 모든 마취약물들은 등세모근에 근육주사를 하였다. 약물 투여 24시간 전부터 절식을 시켰다. 도입 및 회복 시간을 측정하였다. 심박수, 호흡수 체온, $pO_2,\;pCO_2$ 및 pH를 투여 전, 투여 후 5분, 25분, 45분, 65분 및 85분에 측정하였다. 도입시간에서는 ATZ 군이 TZ군에 비하여 더 빠른 도입을 나타내었다 (p<0.01). 회복에 있어서, 흉와자세 시간, 기립시간 및 보행시간은 ATZ군이 TZ군에 비하여 더 긴 시간을 나타내었다 (p<0.01). 심박수, 호흡수, $pO_2,\;pCO_2$, 및 pH는 두 군간에서 유의차가 인정되지 않았다. 그럼에도 불구하고, 체온은 두 군간 유의성 있는 변화가 인정되었다 (p<0.05). 이상의 결과를 종합하여 볼 때에, ATZ군이 TZ군에 비하여 빠른 도입과 긴 마취시간을 나타내었다. 따라서, ATZ 병용은 돼지의 화학적 보정에 유용하게 사용될 수 있을 것으로 판단되었다.

• Asian-Australasian Journal of Animal Sciences
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• 제27권11호
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• pp.1644-1651
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• 2014

Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine ${\beta}$-casein gene locus using a knock-in vector for the ${\beta}$-casein gene locus. We developed the knock-in vector on the porcine ${\beta}$-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine ${\beta}$-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using ${\beta}$-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous ${\beta}$-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.

• Journal of Plant Biotechnology
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• 제32권4호
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• pp.263-268
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• 2005

Porcine epidemic diarrhea virus (PEDV)는 돼지의 급성장염을 유발하여 설사 증상을 일으키는 바이러스이다. 본 연구에서는 고구마 저장뿌리 고발현 sporamin 프로모터 및 CaMV 35S promoter를 이용하여 PEDV 항원단백질을 생산하는 고구마 식물체를 개발하고자 하였다. 형질전환 벡터를 제작하기 위하여 PEDV에서 항원성이 알려진 스파이크 단백질의 일부분을 암호화하는 유전자를 PCR로 합성하였다. 고구마 [Ipomoea batatas (L.) Lam] 율미 품종의 배발생 캘러스를 재료로 하여 Agrobacterium tumefaciens을 매개로 형질전환하였다. 선발배지 (MS medium, 1 mg/L 2,4-D, 100 mg/L kanamycin, and 400 mg/L claforan)에서 배발생 캘러스를 3주 간격으로 4개월 동안 계대배양하여 카나마이신 저항성 캘러스를 선발하였다. 선발된 배발생 캘러스를 호르몬을 제거한 배지로 옮겨 체세포배를 유도하였으며 이후 shoot과 뿌리가 형성되었다. 재분화된 소식물체를 대상으로 PCR 분석으로 카나마이신 저항성 재분화 개체의 50% 이상이 도입 유전자를 가지고 있었으며 이들 식물체를 대상으로 Southern blot 분석하여 PEDV 유전자가 고구마 식물체의 게놈으로 안정적으로 도입되었음을 확인하였다. 형질전환 식물체에서 도입유전자의 발현을 RT-PCR로 분석한 결과 PEDV의 spike 유전자가 높은 수준으로 발현하였다.

• 한국수정란이식학회지
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• 제29권4호
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• pp.339-344
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• 2014

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. Therefore, we applied CRISPR/Cas9 system to generate FoxN1-targeted pig parthenogenetic embryos. Using single guided RNA targeted to pig FoxN1 genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. In results, regardless of the concentrations of vector, the cleavage rate were significantly (p<0.05) decreased ($4ng/{\mu}l$, 51.24%; $8ng/{\mu}l$, 40.88%; and $16ng/{\mu}l$; 45.22%) compared to no injection group (70.44%). The blastocyst formation rates were also decreased in vector injected 3 groups ($4ng/{\mu}l$, 7.96%; $8ng/{\mu}l$, 6.4%; and $16ng/{\mu}l$; 9.04%) compared to no injection group (29.07%). In addition, the blastocyst formation rates between sham injected group (13.51%) and no injection group (29.07%) also showed significant difference (p<0.05). The mutation rates were comparable between groups ($4ng/{\mu}l$, 18.4%; $8ng/{\mu}l$, 12.5%; and $16ng/{\mu}l$; 20.0%). The sequencing analysis showed that blastocysts derived from each group were successfully mutated in FoxN1 loci regardless of the vector concentrations. However, the deletion patterns were higher than the patterns of point mutation and insertion regardless of the vector concentrations. In conclusion, we described that cytoplasmic microinjection of FoxN1-targeted CRISPR/Cas9 vector could efficiently generate transgenic pig parthenogenetic embryos in one-step.

• Asian-Australasian Journal of Animal Sciences
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• 제20권8호
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• pp.1278-1284
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• 2007

Changes in the concentrations of biogenic amines (BAs) in fresh beef, pork, and chicken breast and leg were investigated during storage, and the relationship between the content of volatile basic nitrogen (VBN) and BAs was evaluated. As the storage period increased, the levels of putrescine (PUT), cadaverine (CAD) and tyramine (TYM) increased in all the meat samples, except for TYM in beef (p<0.001). The level of BAs in beef, pork and chicken changed but the extent of these changes was different among the kinds of BAs and meats. Measurement of the VBN content was confirmed as a good index for interpreting the specific BAs content in general, such as PUT, CAD, and TYM, as well as evaluating a meat's freshness during storage. However, the kinds of BAs which can be predicted from the VBN content varied in different meats (p<0.05).

• 한국수정란이식학회지
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• 제27권2호
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• pp.71-80
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• 2012

Biotechnologies for cloning animals and in vitro embryo production have the potential to produce biomedical models for various researches. Especially, pigs are a suitable model for xenotransplantation, transgenic production and various areas of reproductive research due to its physiological similarities to human. However, utilization of in vitro-produced embryos for transfer remains limited. Despite improvement over past few decades, obstacles associated with the production of good quality embryos in vitro still exist which limit the efficiency of cloning. One of major problems includes improper in vitro maturation (IVM) and culture (IVC). Oxidative stress caused from in vitro culture conditions contributes to inadequate IVM and IVC which leads to poor developmental competence of oocytes, failure of fertilization and embryo development. To reduce the oxidative stress, various antioxidants have been used to IVM and IVC system. However, limited information is available on the effects of resveratrol on livestock reproductions. Resveratrol is a polyphenolic natural product and well known as an antioxidant in foods and beverages (e.g. in grapes and red wine). Resveratrol is known to be cardioprotective, anticarcinogenic, anti-inflammatory, antioxidant and antiapoptotic. This paper will review the effects of resveratrol on in vitro maturation of oocytes and embryo development.