• Reproductive and Developmental Biology
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• 제30권2호
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• pp.87-92
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• 2006

The possibility of producing transgenic embryos expressing the green fluorescence protein (GFP) gene have been evaluated after transfer of exogenous gene into the porcine zygote cytoplasm using the intracytoplasm sperm injection (ICSI) as gene delivery method. For DNA binding to sperm heads, 0.05% Triton X-100 or Lipofectin was used. After injection of the sperm bound to DNA by means of Lipofectin or Triton X-100 triturate, the blastocyst formation rates on day 6 were not significantly different from that of ICSI only group (18.8, 19.2 and 25.3%). In terms of GFP expression, more embryos were in GFP form in Triton X-100 group than in Lipofectin group (40.6 vs 36.4%), while percentage of non-mosaic embryos expressing the GFP gene in all blastomere was higher (P<0.05) in Lipofectin group than in Triton X-100 group (4.2 vs 0.9%). ICSI embryos derived from sperm treated with Lipofectin/DNA complex was transferred into 3 recipients and were collected by uterine flushing on days 5, 7 and 15 after embryo transfer, and then GFP expression was observed by a fluorescence microscopy. Over 26% of the collected embryos were normally expressed GFP gene. These results suggest that foreign gene transfer method with DNA bound sperm caused minimal damage to structure of oocytes that can result to full development of porcine embryos. This was confirmed in this study when the embryos that were transferred after ISCI of DNA bound sperm had a normal development and gene expression until preimplantation.

• Reproductive and Developmental Biology
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• 제33권1호
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• pp.19-24
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• 2009

This study was conducted to investigate the specific expression genes in the cloned bovine tissues. Donor cells, cloned tissues were analysed by RAPD-RFLP method. The results were detected three genes (CH-U7B, CH-U7M and CH-U7P) in the cloned fetus. It was found a single copy genes by southern hybridization. Sequence analysis of CH-U7M gene was shown 99% homology to a previously reported EST from a cloned bovine fetus. The putative ORF was encode a protein of hydrophobicity index 0.03. Semi-quantitative RT-PCR by using the CH-LS001 specific primer was remarkably detected in the lung tissue of cloned fetus. Further investigation of these genes may provide one of the key information to explain the early death, abnormal fetus, large off-spring and the low pregnancy rate in the production of cloned bovine.

• 한국수정란이식학회지
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• 제26권1호
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• pp.79-84
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• 2011

This study was performed to identify the differentially methylated region (DMR) and to examine the mRNA expression of the imprinted H19 gene in day 35 of SCNT pig fetuses. The fetus and placenta at day 35 of gestation fetuses after natural mating (Control) or of cloned pig by somatic cell nuclear transfer (SCNT) were isolated from a uterus. To investigate the mRNA expression and methylation patterns of H19 gene, tissues from fetal liver and placenta including endometrial and extraembryonic tissues were collected. The mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. Bisulfite analyses demonstrated that the differentially methylated region (DMR) was located between -1694 bp to -1338 bp upstream from translation start site of the H19 gene. H19 DMR (-1694 bp to -1338 bp) exhibits a normal mono allelic methylation pattern, and heavily methylated in sperm, but not in oocyte. In contrast to these finding, the analysis of the endometrium and/or extraembryonic tissues from SCNT embryos revealed a complex methylation pattern. The DNA methylation status of DMR Region In porcine H19 gene upstream was hypo methylated in SCNT tissues but hypermethylated in control tissues. Furthermore, the mRNA expression of H19 gene in liver, endometrium, and extraembryonic tissues was significantly higher in SCNT than those of control (p<0.05). These results suggest that the aberrant mRNA expression and the abnormal methylation pattern of imprinted H19 gene might be closely related to the inadequate fetal development of a cloned fetus, contributing to the low efficiency of genomic reprogramming.

• Parasites, Hosts and Diseases
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• 제53권6호
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• pp.675-682
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• 2015

The effects of diclazuril on the bursa of Fabricius (BF) structure and secretory IgA (SIgA) expression in chickens infected with Eimeria tenella were examined. The morphology of the BF was observed by hematoxylin and eosin staining, while ultrastructural changes were monitored by transmission electron microscopy. E. tenella infection caused the BF cell volumes to decrease, irregularly arranged, as well as, enlargement of the intercellular space. Diclazuril treatment alleviated the physical signs of damages associated with E. tenella infection. The SIgA expression in BF was analyzed by immunohistochemistry technique. The SIgA expression increased significantly by 350.4% (P<0.01) after E. tenella infection compared to the normal control group. With the treatment of diclazuril, the SIgA was relatively fewer in the cortex, and the expression level was significantly decreased by 46.7% (P<0.01) compared with the infected and untreated group. In conclusion, E. tenella infection in chickens induced obvious harmful changes in BF morphological structure and stimulated the expression of SIgA in the BF. Diclazuril treatment effectively alleviated the morphological changes. This result demonstrates a method to develop an immunological strategy in coccidiosis control.

• Asian-Australasian Journal of Animal Sciences
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• 제7권1호
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• pp.1-17
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• 1994

There are several gene transfer methods available to introduce foreign DNA into animal. The most common method at present is microinjection. However, the overall efficiency of producing practical application of gene transfer technology to livestock species is production of pharmaceuticals. Rare human proteins, which cannot be produced into milk of transgenic animals. Large amount of biologically active protein may be obtained from transgenic farm animals using this system. Growth-related application to livestock species using growth hormone genes or factor genes have been disappointing. There were many undesirable side effects noted in the transgenic animals. More sophisticated on or off transgene expression are needed to control expression of transgenes in the transgenic animals. Turning positive effects while circumventing potentially harmful effects.

• Asian-Australasian Journal of Animal Sciences
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• 제32권2호
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• pp.257-264
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• 2019

Objective: Dairy cattle nutrient requirement systems acknowledge amino acid (AAs) requirements in aggregate as metabolizable protein (MP) and assume fixed efficiencies of MP used for milk protein. Regulation of mammary protein synthesis may be associated with AA input and milk protein output. The aim of this study was to evaluate the effect of nanoemulsified methionine and cysteine on the in-vitro expression of milk protein (casein) in bovine mammary epithelial cells (MAC-T cells). Methods: Methionine and cysteine were nonionized using Lipoid S 75 by high-speed homogenizer. The nanoemulsified AA particle size and polydispersity index were determined by dynamic light scattering correlation spectroscopy using a high-performance particle sizer instrument. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the cytotoxicity effect of AAs with and without nanoionization at various concentrations (100 to $500{\mu}g/mL$) in mammary epithelial cells. MAC-T cells were subjected to 100% of free AA and nanoemulsified AA concentration in Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12) for the analysis of milk protein (casein) expression by the quantitative reverse transcription polymerase chain reaction method. Results: The AA-treated cells showed that cell viability tended to decrease (80%) in proportion to the concentration before nanogenesis, but cell viability increased as much as 90% after nanogenesis. The analysis of the expression of genetic markers related to milk protein indicated that; ${\alpha}_{s2}$-casein increased 2-fold, ${\kappa}$-casein increased 5-fold, and the amount of unchanged ${\beta}$-casein expression was nearly doubled in the nanoemulsified methionine-treated group when compared with the free-nanoemulsified methionine-supplemented group. On the contrary, the non-emulsified cysteine-administered group showed higher expression of genetic markers related to milk protein ${\alpha}_{s2}$-casein, ${\kappa}$-casein, and ${\beta}$-casein, but all the genetic markers related to milk protein decreased significantly after nanoemulsification. Conclusion: Detailed knowledge of factors, such nanogenesis of methionine, associated with increasing cysteine and decreasing production of genetic markers related to milk protein (casein) will help guide future recommendations to producers for maximizing milk yield with a high level of milk protein casein.

• Asian-Australasian Journal of Animal Sciences
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• 제24권2호
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• pp.272-278
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• 2011

Bovine whey protein expression patterns of colostrum are much different from that of milk. Moreover, bovine colostrum is an important source of protective, nutritional and developmental factors for the newborn. However, to our knowledge, no research has been performed to date using a comparative proteomic method on the changes in the bovine whey proteome during the transition from colostrum to milk. This study therefore separated whey protein of days 1, 3, 7 and 21 after calving using two dimension electrophoresis. Differentially expressed proteins at different collection times were identified using high-performance liquid chromatography in tandem with mass spectrometry (LC/MS) and validated by enzyme-linked immunosorbent assay (ELISA) in order to understand the developmental changes in the bovine whey proteome during the transition from colostrum to milk. The expression patterns of whey protein of days 1 and 3 post-partum were similar except that immunoglobulin G was down-regulated on day 3, and four proteins were found to be down-regulated on days 7 and 21 compared with day 1 after delivering, including immunoglobulin G, immunoglobulin M, albumin, and lactotransferrin, which are involved in immunity and molecule transport. The results of this study confirm the comparative proteomic method has the advantage over other methods such as ELISA and immunoassays in that it can simultaneously detect more differentially expressed proteins. In addition, the difference in composition of milk indicates a need for adjustment of the colostrum feeding regimen to ensure a protective immunological status for newborn calves.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.114-115
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• 2005

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.114-115
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• 2005

• Asian-Australasian Journal of Animal Sciences
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• 제25권7호
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• pp.950-955
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• 2012

This study was conducted to determine how the isolation method of the porcine preantral follicles influenced the following follicular growth in vitro. Mechanical and enzymatical isolations were used for retrieving the follicles from prepubertal porcine ovaries, and in vitro-growth of the follicles and the expression of folliculogenesis-related genes were subsequently monitored. The enzymatic retrieval with collagenase treatment returned more follicles than the mechanical retrieval, while the percentage of morphologically normal follicles was higher with mechanical retrieval than with enzymatic retrieval. After 4 days of culture, mechanically retrieved, preantral follicles yielded more follicles with normal morphology than enzymatically retrieved follicles, which resulted in improved follicular growth. The mRNA expression of FSHR, LHR Cx43, DNMT1 and FGFR2 genes was significantly higher after culture of the follicles retrieved mechanically. These results suggest that mechanical isolation is a better method of isolating porcine preantral follicles that will develop into competent oocytes in in vitro culture.