• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.217-217
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• 2004

The present study was conducted to investigate the developmental competency between male- and female-somatic cell derived nuclear-transferred porcine embryos, and the productive and survival efficiency of cloned male and female piglets. The potential of eggs receiving somatic cells to develop into blastocysts was not different among donor cells of different origins. (omitted)

• 한국수정란이식학회지
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• 제9권2호
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• pp.161-166
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• 1994

This experiment was carried out to produce cloned aniraals by nuclear transplantation in rabbits. The ovulated oocytes were collected from the oviducts between 14 and 15 hours after hGG injection. The denuded oocytes were used as nuclear recipient cytoplasm following enucleation by micromanipulation. The blastomeres separated from the 8-cell embryos were used as nuclear donor. The nucleated oocytes receiving a blastomere in the perivitelline space were electrically fused in the 0.28 M mannitol solution at 1.5 kV /cm, 60$\mu$sec for three times. The nuclear transplant embryos which were used and developed to 2- to 4-cell stage in vitro were transferred into the oviducts of synchronized recipient does. A total of 64 nuclear transplant embryos were transferred to 7 recipient does and produced three offspring(4.7%) from a foster mother 31 days after embryo transfer.

• Reproductive and Developmental Biology
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• 제34권3호
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• pp.127-134
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• 2010

This study was to investigate the effect of flavonoid treatment on in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos, and their pregnancy and delivery rate after embryo transfer into recipient. In experiment 1, to optimize the flavonoid concentration, parthenogenetic day 2 ($\geq$ 2-cell) embryos were cultured in 0 (control), 1, 10 and $20\;{\mu}M$ flavonoid for 6 days. In the results, in vitro development rate was the highest in $10\;{\mu}M$ flavonoid group (57.1%) among treatment groups (control, 49.5%; $1\;{\mu}M$, 54.2%; $20\;{\mu}M$, 37.5%), and numbers of total and ICM cells were significantly (p<0.05) higher in $10\;{\mu}M$ flavonoid group than other groups. We found that $10\;{\mu}M$ flavonoid treatment can significantly (p<0.05) decrease the apoptotic index and derive high expression of anti-oxidant, anti-apoptotic, cell growth and development marker genes such as Mn-SOD, Survivin, Bax inhibitor, Glut-5, In-tau, compared to control group. In experiment 2, to produce the cloned Jeju Black Cattle, beef quality index grade 1 bull somatic cells were transferred into enucleated bovine MII oocytes and reconstructed embryos were cultured in $10\;{\mu}M$ flavonoid added medium. When the in vitro produced day 7 or 8 SCNT blastocysts were transferred into a number of recipients, $10\;{\mu}M$ flavonoid treatment group presented higher pregnancy rate (10.2%, 6/59) than control group (5.9%, 2/34). Total three cloned Jeju Black calves were born. Also, two cloned calves in $10\;{\mu}M$ flavonoid group were born and both were all healthy at present, while the one cloned calf born in control group was dead one month after birth. In addition, when the result of short tandem repeat marker analysis of each cloned calf was investigated, microsatellite loci of 11 numbers matched genotype between donor cell and cloned calf tissue. These results demonstrated that the flavonoid addition in culture medium may have beneficial effects on in vitro and in vivo developmental capacity of SCNT embryos and pregnancy rate.

• 한국축산식품학회지
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• 제39권4호
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• pp.601-609
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• 2019

Bifidobacterium longum KACC 91563 secretes family 5 extracellular solute-binding protein via extracellular vesicle. In our previous work, it was demonstrated that the protein effectively alleviated food allergy symptoms via mast cell specific apoptosis, and it has revealed a therapeutic potential of this protein in allergy treatment. In the present study, we cloned the gene encoding extracellular solute-binding protein of the strain into the histidine-tagged pET-28a(+) vector and transformed the resulting plasmid into the Escherichia coli strain BL21 (DE3). The histidine-tagged extracellular solute-binding protein expressed in the transformed cells was purified using Ni-NTA affinity column. To enhance the efficiency of the protein purification, three parameters were optimized; the host bacterial strain, the culturing and induction temperature, and the purification protocol. After the process, two liters of transformed culture produced 7.15 mg of the recombinant proteins. This is the first study describing the production of extracellular solute-binding protein of probiotic bacteria. Establishment of large-scale production strategy for the protein will further contribute to the development of functional foods and potential alternative treatments for allergies.

• Asian-Australasian Journal of Animal Sciences
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• 제16권1호
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• pp.15-22
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• 2003

Experiments were conducted to evaluate the effect of blastomere diameters and cell cycle stages on the subsequent development of nuclear transplant rabbit embryos (NT-embryos) using nuclei derived from the 16- or 32-cell stage embryos. All blastomeres and NT-embryos were cultured individually in modified Ham's F-10 medium supplemented with 10% rabbit serum (RS) at $38^{\circ}C$ and 5% $CO_2$ in air. The diameter of blastomeres from 16-cell stage embryos was found twice of those from 32-cell stage (51 vs 27 ${\mu}m$). Significant differences were observed in cleavage rates ($\geq$3 divisions) in the isolated single blastomeres (54 vs 48 for 16-cell; 28 vs 14 for 32-cell, p<0.05), but the fusion rates of oocytes with transferred nuclei were similar between small and large single blastomeres derived from either 16-cell or 32-cell stage embryos. When 16-cell stage blastomeres were used as nuclear donors, cleavage rates ($\geq$3 divisions) of the NT-embryos were greater in the small nuclear donors than in the large donors (73 vs 55%, p<0.05). On the contrary, significantly higher cleavage (43 vs 6%, p<0.05) and developmental rates (14 vs 0%, p<0.05) were observed in the large blastomere nuclear donor group of the 32-cell stage embryos. When the cell cycle stages were controlled by a microtubule polymerization inhibitor (Demicolcine, DEM) or the combined treatment of DEM and Aphidicolin (APH), a DNA polymerase inhibitor, fusion rates were 88-96% for the 16-cell donor group (without DEM treatment), which were greater than the 32-cell donor group (54-58%). Cleavage rates were also greater in the transplants derived from G1 nuclear donor group (93-95%) than those from the DEM and APH combined treatment (73%) for the 16-cell donor group (p<0.05). No significant difference was detected in the morula/blastocyst rates in either donor cell stage (p>0.05). In conclusion, it appeared that no difference in the developmental competence between large and small isolated blastomeres was observed. When smaller 16-cell stage blastomeres were used as nuclear donor, the cleavage rate or development of NT-embryos was improved and was compromised when 32-cell stage blastomeres were used. Therefore, control nuclear stage of the donor cell at $G_1$ phase in preactivated nuclear recipients seemed to be beneficial for the cleavage rate of the reconstructed embryo in the 16-cell transplant, but not for subsequent morula or blastocyst development.

• 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.

• 생명과학회지
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• 제27권5호
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• pp.524-529
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• 2017

본 연구에서는 zebrafish에 인간의 KCNE1 유전자가 삽입된 형광단백질 vector를 microinjection하고, 그 발현 여부를 확인하고자 하였다. 먼저 양 말단에 제한효소(EcoRΙ, BamHΙ) site를 넣어 제작한 primer들로 genomic DNA에서 KCNE1 유전자를 분리하였다. 그 결과는 약 402 bp 크기의 DNA band였고 이 PCR 산물을 형광단백질 vector인 pPB-CMVp-EF1-GreenPuro 속에 클로닝하여 pPB-CMVp-hKCNE1-EF1-GreenPuro plasmid를 제작하였다. 이렇게 준비된 형광 vector를 zebrafish 수정란에 microinjection하였고, 부화된 치어에서 RT-PCR과 DNA sequencing을 통해 GFP 및 hKCNE1의 발현을 최종 확인하였다. 본 연구는 향후 QT 연장증후군(LQTs)에 대한 동물 모델로써 신경자극 전도, 유전자 치료, 유용 유전자 클로닝을 위한 기술 개발에 응용될 수 있을 것으로 기대된다.

• BMB Reports
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• 제42권5호
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• pp.310-314
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• 2009

Globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) are the proposed functional receptors for Shiga toxin (Stx). To elucidate the effect of Gb3 content on Stx-induced cytotoxicity in HeLa cells, we cloned HeLa cells and determined the correlation between glycolipids content and Stx-induced cytotoxicity. The 29 HeLa cell clone (HLCC) lines used showed a wide range of sensitivity to Stx, compared to Gb3-rich cells which were more sensitive, showing as little as 20% viability to 100 pg/ml Stx. In contrast, Gb3-deficient cells proved resistant as they were more than 80% viable to 100 ng/ml Stx. Gb3 content in the HLCC lines corresponded with Stxs-induced cytotoxicity as well as Gb3 synthase expression, but no correlation with Gb4 content was noted. These data show that Gb3 content, which is regulated by the expression of Gb3 synthase, determines the sensitivity of HeLa cells toward Stx.

• 한국축산식품학회지
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• 제39권1호
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• pp.13-22
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• 2019

This study aimed to develop a bile-responsive expression system for lactobacilli. The promoters of four genes, encoding phosphoenolpyruvate-dependent sugar phosphotransferase (mannose-specific), L-lactate dehydrogenase (LDH), HPr kinase, and D-alanine-D-alanine ligase, respectively, which were highly expressed by bile addition in Lactobacillus johnsonii PF01, were chosen. Each promoter was amplified by polymerase chain reaction and fused upstream of the ${\beta}$-glucuronidase gene as a reporter, respectively. Then, these constructs were cloned into E. coli-Lactobacillus shuttle vector pULP2, which was generated by the fusion of pUC19 with the L. plantarum plasmid pLP27. Finally, the constructed vectors were introduced into L. plantarum for a promoter activity assay. The LDH promoter showed the highest activity and its activity increased 1.8-fold by bile addition. The constructed vector maintained in L. plantarum until 80 generations without selection pressure. A bile-responsive expression vector, $pULP3-P_{LDH}$, for Lactobacillus spp. can be an effective tool for the bile-inducible expression of bioactive proteins in intestine after intake in the form of fermented dairy foods.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.187-187
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• 2004

Gene delivery is one of the keen interests in animal industry as well as research on gene function. 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. (omitted)