• Korean Journal of Environmental Biology
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• v.22 no.2
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• pp.329-335
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• 2004

To develop a biomarker for the monitoring of the contamination of estrogenic endocrine disrupters in the aquatic environment, reverse transcription -polymerase chain reaction (RT-PCR) analysis of vitellogenin (Vg) mRNA expression was optimized in Bombina orientalis, a Korean red bellied toad species. Based on partial cDNA sequences of both Vg and beta actin genes of B. orientalis, specific primers for RT-PCR of Vg and beta actin mRNAs were developed. Semiquantitative RT-PCR of the Vg mRNA in liver was optimized using a beta actin mRNA as an internal control in both sexes. In female RT-PCR using $1\;\mu{g}$ of the liver cDNA resulted in a linear increment in the PCR product of Vg from 18 to 34 cycles of amplification. In male, on the contrary, the RT- PCR product was first detected at 30 cycles of amplification and a linear increment was observed from 30 to 40 cycles of amplification, suggesting that male B. orientalis expresses minute amount of Vg mRNA which is a $2^{-12}$ equivalent of female. In conclusion, the optimized protocol for semiquantitative RT-PCR analysis of Vg mRNA level in B. orientalis male liver will be useful for the environmental monitoring the xenoestrogen contamination in the freshwater environment in Korea.

• Reproductive and Developmental Biology
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• v.31 no.1
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• pp.55-60
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• 2007

Aging causes thymus involution, and genes in thymus play an important role in the development of the immune system. In this study, we compared genes expressed in thymus of neonatal and peripubertal rats using annealing control primers (ACPs)-based GeneFishing polymerase chain reaction (PCR) and semiquantitative reverse transcription (RT)-PCR. We identified 10 differentially expressed genes (DEGs) with 20 ACPs. Of 10 DEGs, bystin-like, collagen type V alpha 1 (COL5A1), and T-cell receptor beta-chain segment 2 (TCRB2) that are related to immune-function were detected in rat thymus. Bystin-like and TCRB2 were up-regulated, while COL5A1 was down-regulated in peripubertal thymus. Semiquantitative RT-PCR confirmed postnatal changes in expression of bystin-like, COL5A1, and TCRB2. These results suggest that bystin-like, COL5A1, and TCRB2 could regulate immune function controlled in thymus as age increases.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.62-62
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• 2003

Aquaporin (AQP) family protein은 일종의 수분 전달 통로 역할을 하는 단백질로 AQP를 통한 수분의 조절은 삼투압을 통한 물의 이동과 함께 조직내 정상적인 수분의 상성 유지에 필수적이다. 현재까지 11종의 AQP이 신장·뇌·정소·안구 등에서 발현이 확인되었다. AQP9은 물 뿐 아니라 carbamide, polyol, purine, pyrimidine, urea, glycerol 등의 이동에 관여한다. 본 연구에서는 생쥐에서 출생 후 성체에 이르는 동안 정소 내 AQP9의 발현, Leydig cell의 분화에 따른 AQP9의 발현을 조사하였다. 1, 2, 4, 8주령의 정소로부터 semiquantitative RT-PCR 및 real time PCR 법으로 AQP9의 발현을 분석한 결과 1주령에서는 발현되지 않았고 2주령에서는 미량이 발현되기 시작하였고, 4주령에서는 성체의 1/2수준으로 발현량이 급격히 증가하였고 성체에서는 다량으로 발현됨이 확인되었다. Semiquantitative RT-PCR 법과 real time PCR법을 비교할 때 주령별 발현 양상은 유사하였으나 4주령과 성체에서는 두 시험법 사이에 양적인 차이가 있었다. 면역조직화학염색 결과 주로 Leydig cell에서 AQP9의 발현이 확인되었다. 성체의 정소 균질액의 Western blot 상에서 분자량 80, 55, 35 및 23 kDa의 항원이 검출되어 dimer, trimer 형태로 존재할 가능성과 당쇄 결합에 의한 단백질의 변형이 있는 것으로 추정된다. 미성숙 개체의 정소에서는 23 form이 확인되는 반면 성체에서는 35 kDa form이 주로 발현되므로 정소에서 발현되는 AQP9의 경우 Post-translation 수준에서 AQP9의 변형이 수반되는 것으로 사료되며 AQP9의 기능과의 연관성은 추후 연구되어야 할 것이다. Leydig cell은 fetal 및 adult type 2종의 세포가 정소발달 과정에 출현, 사멸, 분화하며 이들은 각기 정소발달, 성숙과 정자형성에 필요한 steroidogenesis에 관여한다. 정소 내 AQP9의 발현은 17beta HSD의 발현 양상과 같게 나타나므로 성적 성숙에 따른 정소 내 AQP9의 발현의 증가는 adult type Leydig cell의 분화와 관련된 것으로 추측된다. 성체의 정소로부터 분리한 Leydig cell-enriched culture에 hCG를 처리한 결과 배양체의 AQP9의 발현이 증가하므로 AQP9은 LH 수용체 하위 신호전달과정을 통해 Leydig cell의 steroidogenesis 또는 생성된 steroids의 분비에 요구되는 수분 및 중성용질의 이동에 관여하는 것으로 사료된다.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.1-8
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• 2011

Techniques to evaluate gene expression profiling, such as sufficiently sensitive cDNA microarrays or real-time quantitative PCR, are efficient methods for monitoring human pluripotent stem cell (hESC/iPSC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turn-around time, and the involvement of highly specialized technical expertise. Hence, there is an urgency of rapid, cost-effective, robust, yet sensitive method development for routine screening of hESCs/hiPSCs. A critical requirement in hESC/hiPSC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all three germ layers, including ectoderm, mesoderm, and endoderm. To quantify the modulation of gene expression in hESCs/hiPSC during their propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR platform technology. Among the 9 gene primers tested, 5 were pluripotent markers comprising set 1, and 3 lineage-specific markers were combined as set 2, respectively. We found that these 2 sets were not only effective in determining the relative differentiation in hESCs/hiPSCs, but were easily reproducible. In this study, we used the hES/hiPS cell lines to standardize the technique. This multiplex RT-PCR assay is flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC/hiPSC lines during routine maintenance and directed differentiation.

• Journal of fish pathology
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• v.19 no.3
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• pp.189-195
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• 2006

A subtracted cDNA library of a marine scuticociliate, Philasterides dicentrarchii, in response to 17β-estradiol exposure was constructed using suppression subtractive hybridization (SSH). As a result of SSH, 275 clones were isolated, and among them, only glutathione peroxidase (GPX) gene was isolated as an antioxidative enzyme responding to 17β-estradiol. The semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis revealed that the transcription of GPX gene of P. dicentrarchii was clearly increased by exposure to 17β-estradiol. The GPX transcription was also clearly increased by exposure to xenoestrogens such as bisphenol A (BPA) and genistein.

• Journal of Animal Science and Technology
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• v.44 no.6
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• pp.677-684
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• 2002

We have investigated the expression patterns of candidates for growth stage specifically expressed genes. The expression patterns of the EPV20, aldolase A, Translationally Controlled Tumor Protein (TCTP) and Adipocyte Differentiation Related Protein (ADRP) were examined by semiquantitative RT-PCR and northern blot analysis in skeletal muscle tissues of Hanwoo, especially in the longissimus dorsi at various growth stages. The EPV20 mRNA was expressed in longissimus dorsi tissue of Hanwoo, but there was no difference of expression levels during growth stages. Though the aldolase A gene was reported to be muscle-specific and regulated at developmental stages, the expression levels of aldolase A mRNA in the longissimus dorsi tissues showed little differences at various growth stages. The expression levels of TCTP which was reported as growth-related protein regulated at translation step were gradually increased during growth of Hanwoo. The expression levels of ADRP mRNA were rapidly increased at 24-month-old longissimus dorsi tissue of Hanwoo, and decreased at 30-month-old. Our data suggest that the ADRP gene show as growth-stage dependent expression and is related to fat deposition within muscular tissue.

• IMMUNE NETWORK
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• v.2 no.4
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• pp.195-201
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• 2002

Background: IgE is closely related to the development of allergies. However, the poor relationship between the specific IgE level and the severity of allergic diseases suggests the possibility of functionally different IgE isoforms. With this in mind, rat basophilic leukemia (RBL)-2H3 activation was analyzed with each type of rat IgE for two parameters, exocytosis and IL-4 mRNA production. RBL-2H3 has been well documented in the rat mucosal mast cell line. Methods: RBL-2H3 cells sensitized with each kind of rat IgE was activated by cross-linking FcRI with B5 (monoclonal anti-rat IgE mouse IgG antibodies). The RBL-2H3 exocytosis was measured by analyzing the ${\beta}$-hexosaminidase level, and the level of IL-4 mRNA synthesis was analyzed using semiquantitative RT-PCR. Rat IgE, which was produced by a parasite infection (REP), was prepared using either Paragonimus westermani metacercariae (REP-PW) or Anisakis simplex third stage larvae (REP-AS). A rat IgE prototype of IR162 was prepared by a peritoneal injection of immunocytoma. Results: The level of exocytosis showed a linear relationship with the rat IgE concentration when REP-PW or REP-AS was applied. However, it exhibited a biphasic response with IR162. In addition, the time course of heating at $56^{\circ}C$ illustrated the similarity between REP-PW and REP-AS, which differed from that of IR162. In contrast, the level of IL-4 mRNA synthesis in the RBL-2H3 cells with IR162 was comparable to that of either REP-PW or REP-AS. Conclusion: These results suggest that functionally different rat IgE isoforms exists in RBL-2H3 exocytosis.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.2
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• pp.1017-1021
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• 2013

The cellular apoptosis susceptibility (CSE1L) gene has been demonstrated to regulate multiple cellular mechanisms including the mitotic spindle check point as well as proliferation and apoptosis. However, the importance of CSE1L in human colon cancer is largely unknown. In the present study, we examined expression levels of CSE1L mRNA by semiquantitative RT-PCR. A lentivirus-mediated small interfering RNA (siRNA) was used to knock down CSE1L expression in the human colon cancer cell line RKO. Changes in CSE1L target gene expression were determined by RT-PCR. Cell proliferation was examined by a high content screening assay. In vitro tumorigenesis was measured by colony-formation assay. Cell cycle distribution and apoptosis were detected by flow cytometric analysis. We found CSE1L mRNA to be expressed in human colon cancer cells. Using a lentivirus based RNAi approach, CSE1L expression was significantly inhibited in RKO cells, causing cell cycle arrest in the G2/M and S phases and a delay in cell proliferation, as well as induction of apoptosis and an inhibition of colony growth capacity. Collectively, the results suggest that silencing of CSE1L may be a potential therapeutic approach for colon cancer.

• Molecules and Cells
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• v.19 no.1
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• pp.31-38
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• 2005

Human embryonic stem (hES) cells have unique features including unlimited growth capacity, expression of specific markers, normal karyotypes and an ability to differentiate. Many investigators have tried to use hES cells for cell-based therapy, but there is little information about the properties of available hES cell lines. We compared the characteristics of three hES cell lines. The expression of SSEA-1, -3, -4, and APase, was examined by immunocytochemistry, and Oct-4 expression was analyzed by RT-PCR. Differentiation of the hES cells in vitro and in vivo led to the formation of embryoid bodies (EBs) or teratomas. We examined the expression of tissue-specific markers in the differentiated cells by semiquantitative RT-PCR, and the ability of each hES cell line to proliferate was measured by flow cytometry of DNA content and ELISA. The three hES cell lines were similar in morphology, marker expression, and teratoma formation. However there were significant differences (P < 0.05) between the differentiated cells formed by the different cell lines in levels of expression of tissue-specific markers such as renin, kallikrein, Glut-2, ${\beta}-$ and ${\delta}-globin$, albumin, and ${\alpha}1-antitrypsin$ (${\alpha}1-AT$). The hES cell lines also differed in proliferative activity. Our observations should be useful in basic and clinical hES cell research.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.22-22
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• 2003

This study was conducted to determine effects of polyvinyl alcohol (PVA), fetal bovine serum (FBS) and bovine serum albumin (BSA) on blastocoel formation, cell number, apoptosis and apoptosis-related gene expression of porcine diploid parthenotes developing in vitro. Embryos were collected from 2-cell or late 4-cell diploid parthenotes that activated with electro pulse, and in vitro cultured in the NCSU 23 medium supplemented without or with 0.1% PVA, 10% FBS or 0.4% BSA for day 7. The morphological analysis of apoptosis in embryos was carried out using propidium iodide staining and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The expressions of Bcl-xL, Bak and P53 in blastocyst stage parthenotes and in vivo-derived blastocysts were determined using semiquantitative RT-PCR. The addition of 0.4% BSA to the culture medium enhanced the development of 2- or late 4-cell stage parthenotes to the blastocysts stage (P < 0.01) while FBS decreased the incidence of blastocoel formation. FBS also reduced cell numbers of blastocysts developed from both 2- (P < 0.001) and late 4-cell (P < 0.05) embryos and increased percentage of apoptosis in the blastocysts (P < 0.001). The relative abundance of Bcl-xL mRNA in diploid parthenotes cultured from 2-cell stage in the presence of BSA is similar with that in in vivo derived embryos, but is significantly higher than in parthenotes cultured with FBS, PVA or none protein supplement control. Bak mRNA showed a significant increase at the blastocyst stage in FBS supplement medium. This result suggests that apoptosis related gene expression is significantly affected by protein supplements, which may result in alteration of apoptosis and embryo viability of porcine embryos developing in vitro.