• Development and Reproduction
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• v.5 no.1
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• pp.47-52
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• 2001

Predetermination of sex in livestock of offpring is in great demand and is of critical importance to providing for the most efficient production of the animal ariculture. Such a sexing techlology would also enhance the economy of conventional artificial insemination(AI) and aid the porcine industry. The purpose of this study was to evaluate the efficiency of enriching X-bearing porcine sperm using discontinuous percoll gradients and PCR mefhod. Semen was collected from mature boars of proven fertility center (AI center KimHae). Sperm was leaded on the isotonic discontinuous percoll gradient and then it was centrifuged at 120 ${\times}$ g for 20 minutes. After centrifugation, sperm included in each fraction were recovered (7${\times}$10$^6$ sperms/ml) and then sperm genomic DNA was extractedfor the PCR. SRY gene was used to evaluate the ratio between X and Y sperm in the separated fractions. Ju viro ffrtilization wascarried out by adding the unseparated sperm (control) or separated (experimental poop) to the matured oocytes in TCM-199. Embryos for sex determination were obtained at 2 cell stage and then was used for SRY gene amplification. After centrifugation of discontinuous percoll gradient, the most motile sperm was obtained at 95% fiaction (94.4% ${\pm}$ 5.1%, p < 0.01). The PCR analysis evaluated that 30%, 50% and 65% fractions were Y sperm rich, whereas 80% and 95% fractions were X sperm rich. PCR analysis with each porcineembryo showed that 33.3% of control and 66.7% of experimental group were determined as female embryos. In conclusion, in vitro matured oocytes inseminated with sperms (95% fraction) prepared by percoll gradient centrifugation showed high fertilization rates and female embryos than control sperms.

• Development and Reproduction
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• v.5 no.2
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• pp.107-114
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• 2001

The recent reports that endocrine disruptors(EDs) bring about abnormalities in reproductive organs and functions of invertebrates suggest that mammals be affected by the EDs. The present study examined the influence of 2-bromopropane(2-BP) by looking at the sexes of litters in mouse. The expression of sex-related genes during sex differentiation was also investigated in the fetus of mouse. The male and female mice were infused with 2-BP for 3 weeks before mating. The litters were sexed at the weaning time from the 4 different groups. The sex-related genes were identified by RT-PCR from the fetuses at gestation 10 days. The sequences of the genes were analysed by comparing to those of other animals. The mean numbers of litters survived by the weaning time were slightly reduced in the only group of both female and male mice treated with 2-BP. The female litters were greater than male litters in the only group of female treated with 2-BP. The other groups showed male litters greater than female litters. The sex-related genes, SRY, DAX1, SF1 , and AMH genes were identified and sequenced, showing 416, 466, 326, 389 base pairs, respectively. All of the genes had the homology of 89～90％ with rat and 81～92％ with human within the range of bases identified. They were expressed at the time of sex determination. Therefore, it appears that 2-BP somewhat affects the reproductive activity of adult mouse. Influence of 2-BP on the reproductive function is expected to be studied through the expression of the sex-related genes.

• 대한생식의학회:학술대회논문집
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• 1996.11a
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• pp.69.2-70
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• 1996

• Journal of Genetic Medicine
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• v.12 no.2
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• pp.100-108
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• 2015

Purpose: Conventional methods for the prenatal detection of fetal RhD status involve invasive procedures such as fetal blood sampling and amniocentesis. The identification of cell-free fetal DNA (cffDNA) in maternal plasma creates the possibility of determining fetal RhD status by analyzing maternal plasma DNA. However, some technical problems still exist, especially the lack of a positive control marker for the presence of fetal DNA. Therefore, we assessed the feasibility and accuracy of fetal RHD genotyping incorporating the RASSF1A epigenetic fetal DNA marker from cffDNA in the maternal plasma of RhD-negative pregnant women in Korea. Materials and Methods: We analyzed maternal plasma from 41 pregnant women identified as RhD-negative by serological testing. Multiplex real-time PCR was performed by amplifying RHD exons 5 and 7 and the SRY gene, with RASSF1A being used as a gender-independent fetal epigenetic marker. The results were compared with those obtained by postnatal serological analysis of cord blood and gender identification. Results: Among the 41 fetuses, 37 were RhD-positive and 4 were RhD-negative according to the serological analysis of cord blood. There was 100% concordance between fetal RHD genotyping and serological cord blood results. Detection of the RASSF1A gene verified the presence of cffDNA, and the fetal SRY status was correctly detected in all 41 cases. Conclusion: Noninvasive fetal RHD genotyping with cffDNA incorporating RASSF1A is a feasible, reliable, and accurate method of determining fetal RhD status. It is an alternative to amniocentesis for the management of RhD-negative women and reduces the need for unnecessary RhIG prophylaxis.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.3
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• pp.421-427
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• 2012

The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Milk is presently the most mature system for production of therapeutic proteins from a transgenic animal. Specifically, ${\beta}$-casein is a major component of cow, goat and sheep milk, and its promoter has been used to regulate the expression of transgenic genes in the mammary gland of transgenic animals. Here, we cloned the porcine ${\beta}$-casein gene and analyzed the transcriptional activity of the promoter and intron 1 region of the porcine ${\beta}$-casein gene. Sequence inspection of the 5'-flanking region revealed potential DNA elements including SRY, CdxA, AML-a, GATA-3, GATA-1 and C/EBP ${\beta}$. In addition, the first intron of the porcine ${\beta}$-casein gene contained the transcriptional enhancers Oct-1, SRY, YY1, C/EBP ${\beta}$, and AP-1, as well as the retroviral TATA box. We estimated the transcriptional activity for the 5'-proximal region with or without intron 1 of the porcine ${\beta}$-casein gene in HC11 cells stimulated with lactogenic hormones. High transcriptional activity was obtained for the 5'-proximal region with intron 1 of the porcine ${\beta}$-casein gene. The ${\beta}$-casein gene containing the mutant TATA box (CATAAAA) was also cloned from another individual pig. Promoter activity of the luciferase vector containing the mutant TATA box was weaker than the same vector containing the normal TATA box. Taken together, these findings suggest that the transcription of porcine ${\beta}$-casein gene is regulated by lactogenic hormone via intron 1 and promoter containing a mutant TATA box (CATAAAA) has poor porcine ${\beta}$-casein gene activity.

• Proceedings of the KAIS Fall Conference
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• 2007.05a
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• pp.46-47
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• 2007

본 논문은 소의 모색에 관여하는 Melanocortin I recepter (MCIR) 유전자의 염기서열을 기초로 정확하게 한우와 젖소를 구분할 수 있는 단일 염기 변이를 이용하여 기존의 방법보다 2배 이상으로 빠른 속도로 한우와 젖소를 판별할 수 있는 multiplex PCR 기술에 관한 것이다. 또한, 동 기술에 적용된 기술에는 기존의 방법으로는 동시 판별을 할 수 없었던 소의 암수 구분이 가능하도록 수컷 판별을 위한 표지 유전자인 SRY 유전자를 적용함으로써 한우와 젖소 그리고 압수를 고속으로 동시 판별이 가능하게 되어 젖소가 한우로 둔갑되는 현재의 불법 유통의 문제점을 해결해 주는 새로운 방법이 될 수 있을 것으로 사료된다.

• Journal of the Korean Chemical Society
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• v.53 no.6
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• pp.742-748
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• 2009

In the meat industry, correct breed information in food labeling is required to assure meat quality. Genetic markers provide corroborating evidence to identify breed. We described the development of DNA markers to discriminate between Korean beef cattle (Hanwoo), Holstein, and mixed cow beefs. As most breeds are standardized for coat colour, the melanocortin 1 receptor (MC1R) gene, involved in the regulation of eu/pheomelanins synthesis, has been suggested as marker for breed traceability of products of animal origin. We also designed sex-determining region Y (SRY) gene specific primers for Y chromosome detection. In this study, fragments of MC1R gene and SRY gene were amplified by multiplex-PCR and subjected to digestion by MspA1I restriction endonuclease. Reaction products were analysised by denaturing high performance liquid chromatography (DHPLC). As a result, we identified 6 DHPLC peak types from MC1R gene and SRY gene analysis. DHPLC method showed more sensitive than RFLP method for DNA fragments analysis. Therefore, DHPLC method can apply to identify for Hanwoo, Holstein and mixed beef.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.1
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• pp.17-26
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• 2005

Objective: Preimplantation genetic diagnosis (PGD) is reserved for couples with a risk of transmitting a serious and incurable disease, and hence avoids the undesirable therapeutic abortion. In this study, we evaluated the efficacy of PGD for Duchenne muscular dystrophy (DMD) cases by the fluorescent PCR with polymorphic linked markers and the conventional duplex-nested PCR methods. Methods: Biopsy of one or two blastomeres was done from the embryos fertilized by ICSI on the third day after fertilization. We performed two cases of PGD-DMD by the duplex-nested PCR for the causative mutation loci and the SRY gene on Y chromosome. The triplex fluorescent PCR for the mutation loci, the SRY gene and the polymorphic microsatellite marker on X chromosome was applied for two cases of PGD-DMD. Results: By the duplex-nested PCR, successful diagnosis rate was 95.5% (21/22), but we could not discriminate the female embryos whether normal or carrier in this X-linked recessive disease. However, the triplex fluorescent PCR method showed 100% (27/27) of successful diagnosis rate, and all female embryos (n=17) were distinguished normal (n=10) from carrier (n=7) embryos. Unaffected and normal embryos were transferred into mother's uterus after diagnosis. A healthy normal male was achieved after PGD with the duplex-nested PCR method and a twin, a male and a female, were delivered with triplex fluorescent PCR method. The normality of dystrophin gene was confirmed by amniocentesis and postnatal genetic analysis in all offsprings. Conclusion: The fluorescent PCR with polymorphic marker might be useful in improving the specificity and reliability of PGD for single gene disorders.

• Journal of Life Science
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• v.17 no.3 s.83
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• pp.402-406
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• 2007

This study was performed to develop the molecular marker for sex determination of hare (Lepus coreanus) distributed in Korea which focused on sexual dimorphism between X and Y chromosomal homologous genes, zinc finger-X (ZFX) and -Y (ZFY). The intron 7 regions of ZFX and ZFY genes exhibited differential amplification patterns between male and female hares. The lengths of intron 7 region of ZFX and ZFY genes were 538 and 233-bp, respectively. Especially, the ZFX intron 7 contained a repetitive sequence identified as member of RNA-mediated transposable elements which was similar to CSINE2 commonly found in the rabbit genome. However, it was not present in intron 7 of ZFY gene. The molecular sex typing by polymerase chain reaction (PCR) was also carried out to determine the sex of hare based on difference in lengths between the intron 7 regions of ZFX and ZFY genes. All DNA samples tested had common band amplified from ZFX. However, the male hare DNAs had two distinct bands which amplified from ZFX and ZFY genes, respectively. The results from ZFX-ZFY PCR sex typing were identical to those from phenotypic investigation and from amplification patterns using male-specific sex determining region Y (SRY) gene as well. Finally, this study suggested that the sexual dimorphism between intron 7 regions of ZFX and ZFY could be useful genetic marker to determine sex of hare.

• Transactions of the Korean Society of Mechanical Engineers A
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• v.32 no.4
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• pp.371-377
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• 2008

Our precedent study has reported glass-PDMS (polydimethylsiloxane) based biochip for the gene PCR (polymerase chain reaction). To prevent the contamination of bio sample, the once used biochip must not be used repeatedly. However, the fabrication cost of microheater and microsensor of the biochip was not cheap to use it as a disposable chip. This paper proposes new PCR-chip where the glass substrate integrated with the microheater and microsensor is detachable from the reaction chamber where the sample is injected. That makes it possible to reuse the glass substrate repeatedly. The performance of the proposed detachable PCR-chip was compared with that of the precedent monolithic PCR-chip. The results showed that the SRY (sex determining Y chromosome) gene PCR was successfully performed in the detachable chip compared with the monolithic chip. However, the more efforts to improve the efficiency of surface treatment of PDMS chip are needed to increase the possibility of applying the detachable chip to the detecting of male infertility.