• Journal of Veterinary Clinics
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• v.33 no.5
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• pp.266-269
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• 2016

Cell-free fetal RNA is useful to determine fetal sex and detect other inherent genetic disorders. However, non-invasive fetal sex determination methods using fetal RNA from maternal plasma is not yet well established in studies pertaining to bovine animals. Thus, the aim of this study was to systematically evaluate the presence of the male-specific ZRSR2Y gene transcript in maternal plasma using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assays, and to verify its accuracy, sensitivity, and specificity in determining fetal sex between 30 and 100 days of gestation. Overall accuracy, sensitivity, and specificity of the ZRSR2Y gene transcripts in determining fetal sex were 89.1%, 86.3%, and 100%, respectively. The 30 to 100 days of gestation were further classified into five stages of gestation, and each stage had relatively high accurate, sensitive, and specific results. Overall, these results indicate that the expression of the ZRSR2Y gene can be used for fetal sex determination in bovine animals using circulating cell-free RNA in maternal plasma during early pregnancy.

• Journal of Genetic Medicine
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• v.13 no.2
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• pp.65-71
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• 2016

Cell-free nucleic acids (cf-NAs) originate in trophoblasts and are detected in the maternal plasma. Using innovative bioinformatic technologies such as next-generation sequencing, cf-NAs in the maternal plasma have been rapidly applied in prenatal genetic screening for fetal aneuploidy. Amniotic fluid is a complex and dynamic fluid that provides growth factors and protection to the fetus. In 2001, the presence of cf-NA in amniotic fluid was reported. Amniotic fluid is in direct contact with the fetus and is derived from fetal urine and maternal and fetal plasma. Therefore, these genetic materials have been suggested to reflect fetal health and provide real-time genetic information regarding fetal development. Recently, several studies evaluated the global gene expression changes of amniotic fluid cell-free RNA according to gestational age. In addition, by analyzing the transcriptome in the amniotic fluid of fetal aneuploidy, potential key pathways and novel biomarkers for fetal chromosomal aneuploidy were identified. Here, we review the current knowledge of cell-free RNA in amniotic fluid and suggest future research directions.

• Journal of Embryo Transfer
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• v.28 no.4
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• pp.343-348
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• 2013

Cell-free fetal RNA has been highlighted as useful tools for the fetal sex determination or other genetic inherent disorder. However, there is no knowledge about the sex determination using cell free fetal RNA in bovine field. Thus, the present study aimed to evaluate the presence of transcripts of DDX3Y, USP9Y and ZRSR2Y genes in maternal plasma of pregnant cows to determine the sex of the fetus using real-time quantitative polymerase chain reaction assay, and verify its accuracy, sensitivity and specificity compared with the molecular testing and the calf sex at birth. Transcripts of USP9Y and DDX3Y genes were expressed in the all plasma of males and females both the control group and the experimental group. However, ZRSR2Y gene was matched up with the molecular testing and the true sex in control group and has an overall accuracy of 82.6%, a sensitivity of 75%, and a specificity of 100% in experimental group. Therefore, these results indicated that real time PCR technique, as a noninvasive and cost-efficient method, is possible to determination fetal sex in the bovine species using circulating cell free RNA in maternal plasma and especially ZRSR2Y gene could be a good candidate for the RNA based sex determination work.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.1
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• pp.9-14
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• 1999

Factors affecting gene gun-mediated expression of the human erythropoietin gene were investigated in primary cultured oviduct cells from laying hens. The human erythropoietin gene was transfected by a gene gun method at $1.25{\mu}g$ per dish, and cultured in a synthetic serum-free medium for 72 hrs. The concentration of human erythropoietin mRNA was determined by RNA : RNA solution hybridization. In experiment 1, the effect of changing the shooting pressure of DNA-coated microparticles with nitrogen gas was tested at 20 and $60kgf/cm^2$. The results showed that the erythropoietin mRNA concentration was significantly higher at 60 than $20kgf/cm^2$. In experiment 2, the effects of supplementing the medium with fetal calf serum at 10%, and raising the shooting pressure from 60 to $80kgf/cm^2$ on the cell number and erythropoietin gene expression were examined. Although supplementation with fetal calf serum significantly increased the cell numbes compared with no supplemented controls (p < 0.05), erythropoietin mRNA concentration per $10^3$ cells was not affected. Raising the shooting pressure from 60 to $80kgf/cm^2$ did not affect either of the parameters, In experiment 3, the effect of supplementing ascorbate 2-phosphate at 0.5 mM was tested. The results indicated that the ascorbate supplementation significantly increased the cell number (p < 0.05), and tended to increase erythropoietin mRNA concentration (p < 0.1). Thus, for human erythropoietin gene expression by using the gene gun method, shooting pressure with nitrogen gas should be sufficient at $60kgf/cm^2$ and supplementation with ascorbate phosphate would be useful to enhance not only the cell proliferation but also erythropoietin gene expression.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.3
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• pp.414-421
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• 2014

This study was conducted to determine the effects of saturated long-chain fatty acids (LCFA) on cell proliferation and triacylglycerol (TAG) content, as well as mRNA expression of ${\alpha}s1$-casein (CSN1S1) and genes associated with lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows, and were passaged twice. Then cells were cultured with different levels of palmitate or stearate (0, 200, 300, 400, 500, and 600 ${\mu}M$) for 48 h and fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L). The results showed that cell proliferation tended to be increased quadratically with increasing addition of stearate. Treatments with palmitate or stearate induced an increase in TAG contents at 0 to 600 ${\mu}M$ in a concentration-dependent manner, and the addition of 600 ${\mu}M$ was less effective in improving TAG accumulation. The expression of acetyl-coenzyme A carboxylase alpha, fatty acid synthase and fatty acid-binding protein 3 was inhibited when palmitate or stearate were added in culture medium, whereas cluster of differentiation 36 and CSN1S1 mRNA abundance was increased in a concentration-dependent manner. The mRNA expressions of peroxisome proliferator-activated receptor gamma, mammalian target of rapamycin and signal transducer and activator of transcription 5 with palmitate or stearate had no significant differences relative to the control. These results implied that certain concentrations of saturated LCFA could stimulate cell proliferation and the accumulation of TAG, whereas a reduction may occur with the addition of an overdose of saturated LCFA. Saturated LCFA could up-regulate CSN1S1 mRNA abundance, but further studies are necessary to elucidate the mechanism for regulating milk fat and protein synthesis.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.335-339
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• 2003

The aim of this study is to optimize the monolayer cultivation of human articular chondrocytes in serum-free media. For this purpose, chondrocytes were isolated from human articular cartilage and monolayer cultures were performed in DMEM/F12 medium with 10% fetal bovine serum (FBS) or serum-free media (SFM) containing various supplements and epidermal growth factor (EGF). Western blotting analysis, RT-PCR, dimethylmethylene blue (DMB) assay were carried out to evaluate the synthesis of collagen type II (Col. II) and glycosaminoglycans (GAGs). We observed that SFM with EGF stimulated the cell growth while the amounts of synthesized GAGs and Col. II were decreased gradually. However, the Col. II mRNA level was increased when the SFM was replaced by media containing 10% FBS. This study suggests that it is possible to obtain large amount of human articular chondrocytes by short-term monolayer cultures in SFM.