• Journal of Animal Science and Technology
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• v.63 no.2
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• pp.281-294
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• 2021

Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore, the handmade cloning (HMC) technique has been suggested to simplify and advance the cloning process, however, HMC wastes many oocytes and leads to mitochondrial heteroplasmy. To solve these problems, we propose a modified handmade cloning (mHMC) technique that uses simple laboratory equipment, i.e., a Pasteur pipette and an alcohol lamp, applying it to porcine embryo cloning. To validate the application of mHMC to pig cloning, embryos produced through SCNT and mHMC are compared using multiple methods, such as enucleation efficiency, oxidative stress, embryo developmental competence, and gene expression. The results show no significant differences between techniques except in the enucleation efficiency. The 8-cell and 16-cell embryo developmental competence and Oct4 expression levels exhibit significant differences. However, the blastocyst rate is not significantly different between mHMC and SCNT. This study verifies that cloned embryos derived from the two techniques exhibit similar generation and developmental competence. Thus, we suggest that mHMC could replace SCNT for simpler and cheaper porcine cloning.

• The Plant Pathology Journal
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• v.30 no.4
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• pp.437-439
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• 2014

A fungus Pyrenophora tritici-repentis induces tan spot of wheat which is a foliar disease that causes yield loss to wheat crops worldwide. In this study, a new, simple and non-costly technique was performed to produce the sexual stage of this fungus in culture, within 9 weeks using wheat straw. This protocol will be helpful to researchers studying the biology of sexual stage development, disease epidemiology and genetics of this fungus.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.295.1-295.1
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• 2003

A suitable model for the estimation of the drug release from nanoparticles has been varied and problematic, especially for the release from lipid nanoparticles containing water-insoluble drugs, due to the difficult particle collection from the release medium. Dialysis membrane has been widely used for the release test from colloidal carrier systems. The amount of drug from the carriers in normal dialysis diffusion technique was very low typically. (omitted)

• IMMUNE NETWORK
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• v.3 no.3
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• pp.219-226
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• 2003

Background: Phage display is the most widely used technique among display methods to produce monoclonal antibody fragment with a specific binding activity. Having a large library for efficient antibody display/selection is quite laborious process to have more than $10^9$ members of transformants. To overcome these limitations, several in vitro selection approaches have been reported. Ribosome display that links phenotypes, proteins, directly to genotype, mRNA, is one of the in vitro display methods. Ribosome display can reach the size of scFv library up to $10^{14}$ molecules and it can be further diversified during PCR steps. To select the high affinity scFv from one pot library, we established ribosome display technique by modifying the previously reported eukaryotic translation system. Methods: To establish the antibody selection system by ribosome display, we used 3D8, anti-DNA antibody. A 3D8 scFv was synthesized in vitro by an in vitro transcription-translation system. The translated 3D8 scFv and the encoding 3D8 mRNA are connected to the ribosome. These scFv-ribosome-mRNA complexes were selected by binding to their specific antigens. The eluted mRNAs from the complexes are reverse transcribed and re-amplified by PCR. To apply this system, antibody library from immunized mouse with terminal protein (TP)-peptide of hepatitis B virus DNA polymerase TP domain was also used. This TP-peptide encompasses the 57~80 amino acid residues of TP. These mRNA/ribosome/scFv complexes by our system were panned three times against TP-peptide. The enrichment of antibody from library was determined by radioimmunoassay. Results: We specifically selected 3D8, anti-DNA antibody, against ssDNA as a model system. The selected 3D8 RNAs sequences from translation complexes were recovered by RT-PCR. By applying this model system, we enriched TP-peptide-specific scFv pools through three cycles of panning from immunized library. Conclusion: We show that our translating ribosome complexes are well maintained and we can enrich the TP-specific scFv pools. This system can be applied to select specific antibody from an antibody library.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.305-313
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• 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.

• Proceedings of the KOSOMBE Conference
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• v.1992 no.11
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• pp.39-42
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• 1992

Color Doppler flow mapping (CDFM) was performed on an $\underline{in\;vitro}$ experimental setup with a regurgitant moving orifice using the proximal isovelocity surface area (PISA) technique. PISA flow rates underestimated actual flow rates by as much as 65%, which is very important in diagnosing patients with valvular regurgitations or stenosis. The correction factor considering the velocity of the orifice improved the PISA flow rates.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.10
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• pp.1404-1410
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• 2012

In vitro experiments were conducted to evaluate the suitability of several mixtures of high tanniniferous non legumes with low tanniniferous legumes on in vitro gas production (IVGP), dry matter degradation, Ammonia-N, methane production and microbial population. Eight treatments were examined in a randomized complete block design using four non-legumes and two legumes (Carallia integerrima${\times}$Leucaena leucocephala (LL) (Trt 1), C. integerrima${\times}$Gliricidia sepium (GS) (Trt 2), Aporosa lindeliyana${\times}$LL (Trt 3), A. lindeliyana${\times}$GS (Trt 4), Ceiba perntandra${\times}$LL (Trt 5), C. perntandra${\times}$GS (Trt 6), Artocarpus heterophyllus${\times}$LL (Trt 7), A. heterophyllus${\times}$GS (Trt 8). The condensed tannin (CT) content of non legumes ranged from 6.2% (Carallia integerrima) to 4.9% (Ceiba perntandra) while the CT of legumes were 1.58% (Leucaena leucocephala) and 0.78% (Gliricidia sepium). Forage mixtures contained more than 14% of crude protein (CP) while the CT content ranged from 2.8% to 4.0% respectively. Differences (p<0.05) were observed in in vitro gas production (IGVP) within treatments over a 48 h period dominated by C. perntandra${\times}$G. sepium (Trt 6). The net gas production (p<0.05) was also high with Trt6 followed by A. heterophyllus${\times}$L. leucocephala (Trt 7) and A. heterophyllus${\times}$G. sepium (Trt 8). Highest (p>0.05) NH3-N (ml/200 mg DM) production was observed with the A. heterophyllus${\times}$G. sepium (Trt 8) mixture which may be attributed with it's highest CP content. The correlation between IVGP and CT was 0.675 while IVGP and CP was 0.610. In vitro dry matter degradation (IVDMD) was highest in Trt 8 as well. Methane production ranged from 2.57 to 4.79 (ml/200 mg DM) to be synonimous with IVGP. A higher bacteria population (p<0.05) was found in C. perntandra${\times}$G. sepium (Trt 6) followed by Artocarpus heterophyllus+G. sepium (Trt 8) and the same trend was observed with the protozoa population as well. The results show that supplementing high tannin non leguminous forages by incremental substitution of legume forage increased gas production parameters, NH3-N, IVDMD and microbial population in the fermentation liquid. Methane production was not significantly affected by the presence of CT or different levels of CP in forage mixtures. Among non legumes, Ceiba perntandra and Artocarpus heterophyllus performed better in mixture with L. leucocephala and G. sepium.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.4
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• pp.543-549
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• 2007

Two experiments were conducted to investigate the effects of disodium fumarate on the in vitro rumen fermentation profiles of different substrates and microbial communities. In experiment 1, nine diets (high-forage diet (forage:concentrate, e.g. F:C = 7:3, DM basis), medium-forage diet (F:C = 5:5, DM basis), low-forage diet(F:C = 1:9, DM basis), cracked corn, cracked wheat, soluble starch, tall elata (Festuca elata), perennial ryegrass and rice straw) were fermented in vitro by rumen microorganisms from local goats. The results showed that during 24 h incubations, for all substrates, disodium fumarate increased (p<0.05) the gas production, and tended to increase (p<0.10) the acetate, propionate and total VFA concentration and decrease the ratio of acetate to propionate, whereas no treatment effect was observed for the lactate concentration. The apparent DM loss for tall elata, perennial ryegrass and rice straw increased (p<0.05) with the addition of disodium fumarate. With the exception of tall elata, perennial ryegrass and rice straw, disodium fumarate addition increased the final pH (p<0.05) for all substrates. In experiment 2, three substrates (a high-forage diet, a medium-forage diet and a high concentrate diet) were fermented by mixed rumen microbes in vitro. A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was applied to compare microbial DNA fingerprints between substrates at the end of 24 h incubation. The results showed that when Festuca elata was used as substrate, the control and disodium fumarate treatments had similar DGGE profiles, with their similarities higher than 96%. As the ratio of concentrate increased, however, the similarities in DGGE profiles decreased between the control and disodium fumarate treatment. Overall, these results suggest that disodium fumarate is effective in increasing the pH and gas production for the diets differing in forage: concentrate ratio, grain cereals and soluble starch, and in increasing dry matter loss for the forages (tall elata, perennial ryegrass and rice straw) in vitro, whereas its effect on changes of ruminal microbial community may largely depend on the general nature of the substrate.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.19 no.6
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• pp.147-153
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• 2018

PLGA microcapsule particles encapsulating BSA aqueous solutions were prepared using a water-in-oil-in-water emulsion method. The morphology, particle size, BSA encapsulating efficiency, and in-vitro release test were also studied using the microcapsule particles. In the outer aqueous phase, an emulsifier, e.g., PVA, was replaced with metal salts for surface solidification. Scanning electron microscopy (SEM) showed that the microcapsule particles had smooth surfaces and were between $1{\mu}m$ and $7{\mu}m$ in size. The microcapsule particle morphology was affected directly by the ratio between the polymer solution and inner aqueous solution, and composition of the outer aqueous solution. The factors also partially affected the BSA encapsulation efficiencies and in-vitro release rates. All the microcapsule particles showed an initial burst release through the in-vitro release test. On the other hand, the particles also showed a relatively long release period. Metal salts could be good choices to replace the emulsifier to solidify the microcapsule particle surfaces.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.3
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• pp.325-334
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• 2012

The objective of this study was to determine the roughage to concentrate (R:C) ratio with rain tree pod meal (RPM) supplementation on in vitro fermentation using gas production technique. The experiment design was a 6${\times}$4 factorial arrangement in a CRD. Factor A was 6 levels of R:C ratio (100:0, 80:20, 60:40, 40:60, 20:80 and 0:100) and factor B was 4 levels of RPM (0, 4, 8 and 12 mg). It was found that gas kinetic, extent rate (c) was linearly increased (p<0.01) with an increasing level of concentrate while cumulative gas production (96 h) was higher in R:C of 40:60. In addition, interaction of R:C ratio and RPM level affected $NH_3-N$ and IVDMD and were highest in R:C of 0:100 with 0, 4 mg of RPM and 40:60 with 8 mg of RPM, respectively. Moreover, interaction of R:C ratio and RPM level significantly increased total volatile fatty acids and propionate concentration whereas lower acetate, acetate to propionate ratios and $CH_4$ production in R:C of 20:80 with 8 mg of RPM. Moreover, the two factors, R:C ratio and RPM level influenced the protozoal population and the percentage of methanogens in the total bacteria population. In addition, the use of real-time PCR found that a high level of concentrate in the diet remarkably decreased three cellulolytic bacteria numbers (F. succinogenes, R. flavefaciens and R. albus). Based on this study, it is suggested that the ratio of R:C at 40:60 and RPM level at 12 mg could improve ruminal fluid fermentation in terms of reducing fermentation losses, thus improving VFA profiles and ruminal ecology.