• Journal of Plant Biotechnology
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• 제48권1호
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• pp.34-43
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• 2021

CRISPR/Cas9에 의한 유전자 교정 기술은 유용 형질을 갖는 작물 및 임목의 육성에 있어 널리 사용되고 있는 핵심 기술이다. 유전자 교정 임목 육성에는 아그로박테리움에 의한 형질전환 방법이 높은 효율로 시행된 연구가 많았고 따라서 형질전환에 사용된 플라스미드 서열이 식물 유전체 안에 존재한다는 문제가 남아 있었다. 본 연구에서는 CRISPR/Cas9을 사용하여 유전자 교정 임목을 육성하는 데 기존에 알려진 벡터 도입 기술이 아닌, 단일 가닥 가이드 RNA (sgRNA)와 Cas9 단백질을 혼합하여 만든 리보핵산단백질을 현사시나무 원형질체에 도입하는 방법을 기술하였다. 염 스트레스 내성 관련 인자 PagSAP1 유전자를 표적으로 하는 3종류의 sgRNA를 디자인하고, 각 sgRNA와 Cas9 단백질을 혼합하여 만든 리보핵산단백질을 원형질체에 도입하였다. 표적화 딥시퀀싱을 통해 리보핵산단백질 형성 시 sgRNA와 Cas9 단백질을 혼합하고 일정 시간 배양하여 안정화되는 시간이 필요한 것을 확인하였다. 또한 sgRNA3의 리보핵산단백질이 sgRNA1, sgRNA2의 리보핵산단백질보다 높은 교정 효율을 보이는 것을 확인하였다. 본 실험을 통해 리보핵산단백질을 이용한 유전자 교정 기술이 임목에도 적용될 수 있음이 확인되었고, 이는 외래 유전자 없이 유전자 교정 임목을 육성하는 데 활용할 수 있을 것으로 사료된다.

• Journal of Veterinary Science
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• 제21권2호
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• pp.26.1-26.14
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• 2020

Pancreatic ductal adenocarcinoma is a lethal cancer type that is associated with multiple gene mutations in somatic cells. Genetically engineered mouse is hardly applicable for developing a pancreatic cancer model, and the xenograft model poses a limitation in the reflection of early stage pancreatic cancer. Thus, in vivo somatic cell gene engineering with clustered regularly interspaced short palindromic repeats is drawing increasing attention for generating an animal model of pancreatic cancer. In this study, we selected Kras, Trp53, Ink4a, Smad4, and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) and Streptococcus pyogens Cas9 (SpCas9) for developing pancreatic cancer using adeno associated virus (AAV) transduction. After confirming multifocal and diffuse transduction of AAV2, we generated SpCas9 overexpression mice, which exhibited high double-strand DNA breakage (DSB) in target genes and pancreatic intraepithelial neoplasia (PanIN) lesions with two AAV transductions; however, wild-type (WT) mice with three AAV transductions did not develop PanIN. Furthermore, small-sized Cjcas9 was applied to WT mice with two AAV system, which, in addition, developed high extensive DSB and PanIN lesions. Histological changes and expression of cancer markers such as Ki67, cytokeratin, Mucin5a, alpha smooth muscle actin in duct and islet cells were observed. In addition, the study revealed several findings such as 1) multiple DSB potential of AAV-CjCas9, 2) peri-ductal lymphocyte infiltration, 3) multi-focal cancer marker expression, and 4) requirement of > 12 months for initiation of PanIN in AAV mediated targeting. In this study, we present a useful tool for in vivo cancer modeling that would be applicable for other disease models as well.

• Journal of Ginseng Research
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• 제47권2호
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• pp.337-346
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• 2023

Background: Ginsenoside Rb2, a major active component of Panax ginseng, has various physiological activities, including anticancer and anti-inflammatory effects. However, the mechanisms underlying the rejuvenation effect of Rb2 in human skin cells have not been elucidated. Methods: We performed a senescence-associated β-galactosidase staining assay to confirm cellular senescence in human dermal fibroblasts (HDFs). The regulatory effects of Rb2 on autophagy were evaluated by analyzing the expression of autophagy marker proteins, such as microtubule-associated protein 1A/1B-light chain (LC) 3 and p62, using immunoblotting. Autophagosome and autolysosome formation was monitored using transmission electron microscopy. Autophagic flux was analyzed using tandem-labeled GFP-RFP-LC3, and lysosomal function was assessed with Lysotracker. We performed RNA sequencing to identify potential target genes related to HDF rejuvenation mediated by Rb2. To verify the functions of the target genes, we silenced them using shRNAs. Results: Rb2 decreased β-galactosidase activity and altered the expression of cell cycle regulatory proteins in senescent HDFs. Rb2 markedly induced the conversion of LC3-I to LC3-II and LC3 puncta. Moreover, Rb2 increased lysosomal function and red puncta in tandem-labeled GFP-RFP-LC3, which indicate that Rb2 promoted autophagic flux. RNA sequencing data showed that the expression of DNA damage-regulated autophagy modulator 2 (DRAM2) was induced by Rb2. In autophagy signaling, Rb2 activated the AMPK-ULK1 pathway and inactivated mTOR. DRAM2 knockdown inhibited autophagy and Rb2-restored cellular senescence. Conclusion: Rb2 reverses cellular senescence by activating autophagy via the AMPK-mTOR pathway and induction of DRAM2, suggesting that Rb2 might have potential value as an antiaging agent.

• Molecules and Cells
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• 제24권3호
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• pp.323-328
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• 2007

SOX (Sry-related HMG box) family proteins, which have an evolutionarily conserved DNA binding domain, have crucial roles in cell differentiation. However, their target genes remain enigmatic. Some members of the SOX family may have roles in regulation of cell proliferation. We established stable NT2/D1 cell lines overexpressing SOX15 (SOX15-NT2/D1), and a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the SOX15-NT2/D1 cells exhibited significantly slower growth than the controls. Flow cytometry analysis revealed that an increased fraction of the SOX15-NT2/D1 cells were in G1-G0. In addition, a microarray analysis identified 26 genes that were up-regulated in the SOX15-NT2/D1 cells, but none that were down-regulated genes. Among the up-regulated genes, IGFBP5, S100A4, ID2, FABP5, MTSS1, PDCD4 have been shown to be related to cell proliferation and/or the cell cycle.

• 동의생리병리학회지
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• 제17권1호
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• pp.85-90
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• 2003

We investigated the effects of Oak smoke flavoring (OSF, Holyessing) on the growth of DU145 and PC-3 human prostate carcinoma cells. OSF treatment resulted in a concentration-dependent growth inhibition in both DU145 and PC3 cell lines. The anti-proliferative effect of OSF treatment was associated with the induction of apoptotic cell death which was confirmed by morphological change such as membrane shrinking, rounding up and chromatin condensation in DU145 and PC-3 cells. DNA flow cytometry analysis confirmed that OSF treatment increased population of apoptotic sub-G1 phase. Furthermore, we observed an increase of pro-apoptotic protein Bax expression and a decrease of anti-apoptotic protein Bcl-2 by OSF treatment in a dose-dependent manner. OSF also induced a proteolytic cleavage of specific target proteins such as poly(ADP-ribose) polymerase (PARP) and β-catenin proteins. The present results indicated that OSF-induced inhibition of human prostate carcinoma cell proliferation is associated with the induction of apoptosis.

• 대한임상검사과학회지
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• 제41권1호
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• pp.24-30
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• 2009

Rifampicin (RIF) and isoniazid (INH) are the most important drug for the treatment of Mycobacterium tuberculosis. Mutations correlated to rifampicin and isoniazid-resistance have been detected in rpoB gene and katG gene, respectively. Of the rifampicin-resistant isolates, 90% showed mutations in rpoB gene at codon 507 to 533. Isoniazid-resistant isolates analysed had a mutation in katG at codon 315. The aim of this study is to develop a pyrosequencing-based approach for rapid detection of ripampin or isoniazid resistant M. tuberculosis based on characterization of all possible mutation in the target region. For this study, the DNA selected from 35 cases of MTB PCR positive clinical sample such as bronchial washing, sputum, and pleural fluid. RIF or INH resistant was analyzed by pyrosequencing data of rpoB and katG gene. 28 (80%) and 7 (20%) of 35 MTB PCR positive DNAs were occured rifampicin-sensitivity and resistant, respectively. For INH, 30 (85.7%) and 5 (14.5%) cases were detected isoniazid-sensitivity and resistant, respectively. When pyrosequencing analysis was compared with ABI sequencing analysis, both analysis were presented same result, but pyrosequencing analysis was more rapid than ABI sequencing analysis. In conclusion, we found that pyrosequencing technology offers high accuracy, specificity, short turn around time and a high throughput in detection of rifampicin or isoniazid resistance in M. tuberculosis.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권4호
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• pp.319-324
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• 2006

Although the sera used in animal cell culture media provide the macromolecules, nutrients, hormones, and growth factors necessary to support cell growth, it could also be an obstacle to the production of recombinant proteins in animal cell culture systems used in many sectors of the biotechnology industry. For this reason, many research groups, including our laboratory, have been trying to develop serum-free media (SFM) or serum-supplemented media (SSM) for special or multi-purpose cell lines. The Chinese hamster ovary (CHO) cell, for example, is frequently used to produce proteins and is especially valuable in the large-scale production of pharmaceutically important proteins, yet information about its genome is lacking. Also, SFMs have only been evaluated by comparing growth patterns for cells grown in SFMs with those grown in SSM or by measuring the titer of the target protein obtained from cells grown in each type of medium. These are not reliable methods of obtaining the type of information needed to determine whether an SFM should be replaced with an SSM. We carried out a cDNA microarray analysis to evaluate MED-3, an SFM developed in our laboratory, as a CHO culture medium When CHO cells were cultured in MED-3 instead of an SSM, several genes associated with cell growth were down-regulated, although this change diminished over time. We found that the insulin-like growth factor (IGF) gene was representative of the proteins that were down-regulated in cells cultured in MED-3. When several key supplements - including insulin, transferrin, ethanolamine, and selenium - were removed from MED-3, the IGF expression was consistently down- regulated and cell growth decreased proportionately. Based on these results, we concluded that when an SFM is used as a culture medium, it is important to supplement it with substances that can help the cells maintain a high level of IGF expression. The data presented in this study, therefore, might provide useful information for the design and development of SFM or SSM, as well as for the design of genome-based studies of CHO cells to determine how they can be used optimally for protein production in pharmaceutical and biomedical research.

• Neonatal Medicine
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• 제25권1호
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• pp.49-52
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• 2018

Arterial tortuosity syndrome (ATS) is a very rare autosomal recessive connective tissue disease characterized by generalized elongation and tortuosity of the medium- to large-sized arteries, and other systemic connective tissue manifestations. To date, this disease entity has not been reported in Korea. We report a case of ATS diagnosed in a neonate who presented with severe elongation and tortuosity of the aorta and its major branches, as well as the intracranial arteries. Additionally, the patient presented with a tortuous dilatation of the inferior vena cava, an aneurysmal dilatation of the extra-hepatic bile ducts, and an inguinal and sliding hiatal hernia. The diagnosis was confirmed using DNA sequencing analysis, and the patient demonstrated a compound heterozygosity for two novel mutations (c.738delG [p.Gln247Serfs*33] and c.362T>C [p.Ile121Thr]) in exon 2 of the SLC2A10 gene. Genetic analysis also confirmed that both parents were heterozygous carriers of the responsible mutations. Owing to such clinical manifestations, ATS is often misdiagnosed as other connective tissue diseases including Loeys-Dietz syndrome, Marfan syndrome, and Ehlers-Danlos syndrome. In patients presenting with a high index of suspicion, thorough clinical evaluation and screening for ATS including computed tomography or magnetic resonance angiography and target gene analysis are necessary for early diagnosis and management.

• Bioinformatics and Biosystems
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• 제1권1호
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• pp.28-37
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• 2006

Recent studies in molecular biology and proteomics have identified a significant number of novel diagnostic, prognostic, and therapeutic disease markers. However, validation of these markers in clinical specimens with traditional histopathological techniques involves low throughput and is time consuming and labor intensive. Tissue microarrays (TMAs) offer a means of combining tens to hundreds of specimens of tissue onto a single slide for simultaneous analysis. This capability is particularly pertinent in the field of cancer for target verification of data obtained from cDNA micro arrays and protein expression profiling of tissues, as well as in epidemiology-based investigations using histochemical/immunohistochemical staining or in situ hybridization. In combination with automated image analysis, TMA technology can be used in the global cellular network analysis of tissues. In particular, this potential has generated much excitement in cardiovascular disease research. The following review discusses recent advances in the construction and application of TMAs and the opportunity for developing novel, highly sensitive diagnostic tools for the early detection of cardiovascular disease.

• 생물정신의학
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• 제8권1호
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• pp.106-110
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• 2001

Background : Dopamine receptors have been regarded as a strong candidate involved in etiology of schizophrenia and a target for various antipsychotic drugs. The purpose of our study was to investigate whether dopamine $D_1$ receptor(DRD1) gene polymorphisms would predict the treatment response to antipsychotics in schizophrenia. Method : One hundred thirty-four schizophrenic patients, who met DSM-IV criteria for schizophrenia were entered into a 48 -week study. The psychopathology of the patients was assessed at baseline, 12th, 24th 48th weeks of treatment by PANSS. Responders were defined by a 20% of the reduction in total PANSS score at end point. The genomic DNA fragment corresponding to nucleotides of dopamine $D_1$ receptor gene was amplified by polymerase chain reaction(PCR). Result: Neither allelic frequencies nor genotypes for dopamine $D_1$ receptor differed significantly between responders and non-responders. Also, there was no difference of changes of PANSS scores among three genotype groups of the dopamine $D_1$ receptor. Conclusion : Allelic variation in the dopamine $D_1$ gene is not associated with individual differences in antipsychotic response.