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- 저자 : 이영희 김영은 박정아 (검색결과 6 건)
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Enhancement of Transgene Expression by HDAC Inhibitors in Mouse Embryonic Stem Cells
김영은,박정아,박상규,Ho-Bum Kang,권형주,이영희 한국발생생물학회 2013 발생과 생식 Vol.17 No.4
Embryonic stem (ES) cells can self-renew and differentiate to various cells depending on the culture condition. Although ES cells are a good model for cell type specification and can be useful for application in clinics in the future, studies on ES cells have many experimental restraints including low transfection efficiency and transgene expression. Here, we observed that transgene expression after transfection was enhanced by treatment with histone deacetylse (HDAC) inhibitors such as trichostatin A, sodium butyrate, and valproic acid. Transfection was performed using conventional transfection reagents with a retroviral vector encoding GFP under the control of CMV promoter as a reporter. Treatment of ES cells with HDAC inhibitors after transfection increased population of GFP positive cells up to 180% compared with untreated control. ES cells showed normal expression of stem cell markers after treatment with HDAC inhibitors. Transgene expression was further enhanced by modifying transfection procedure. GFP positive cells selected after transfection were proved to have the stem cell properties. Our improved protocol for enhanced gene delivery and expression in mouse ES cells without hampering ES cell properties will be useful for study and application of ES cells.
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김영은,이영희,박정아,Yang-Wha Ha,박상규,Hee Sun Kim,Sun Kyung Oh 한국발생생물학회 2012 발생과 생식 Vol.16 No.4
Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery. However, human ES cells are difficult to culture and maintain at a large scale, which is one of the most serious obstacles in human ES cell research. Culture of human ES cells on MEF cells after disassociation with accutase has previously been demonstrated by other research groups. Here, we confirmed that human ES cells (H9) can maintain stem cell properties when the cells are passaged as single cells under a feeder-free culture condition. Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression,and morphology. We prepared frozen stocks during the culture period, thawed two of the human ES cell stocks, and analyzed the cells after culture with the same method. Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes. Therefore, we suggest that accutase-dissociated single cells can be usefully expanded in a feeder-free condition but chromosomal modification should be considered in the culture after freeze-thawing.
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OCT4와 SOX2에 의한 인간 Nanog 유전자의 전사 조절
석현정,박정아,이영희,김영은 한국발생생물학회 2010 발생과 생식 Vol.14 No.2
Embryonic stem (ES) cells can self-renew maintaining the undifferentiated state. Self-renewal requires many factors such as OCT4, SOX2, and NANOG. It is previously known that OCT4 and SOX2 can bind to NANOG promoter and support Nanog gene expression in mouse ES cells by the detailed studies using the mouse Nanog promoter. Here, we constructed serial deletion mutant promoter-reporter constructs to investigate the human Nanog gene promoter in detail. The highest promoter activity was obtained in the 0.6 kb (—253/+365) promoter-reporter construct which includes the binding sites of OCT4 and SOX2. To further confirm contribution of OCT4 and SOX2 in Nanog gene expression, we introduced site- directed mutation(s) in the OCT4 and/or SOX2 binding sites of the human Nanog promoter 0.6 kb (—253/+365) and checked the influence of the mutation on the promoter activity using human EC cell line NCCIT. Mutation either in OCT4 binding site or SOX2 binding site significantly reduced the activity of Nanog promoter which directly confirmed that OCT4 and SOX2 binding is essential in human Nanog gene expression.
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히스톤 메틸화 변형을 통한 배아줄기세포의 후성 유전학적 조절
하양화,김영은,박정아,박상규,이영희 한국발생생물학회 2011 발생과 생식 Vol.15 No.4
후성유전학적 조절은 DNA 서열상의 변화 없이도 유전자의 기능을 변화시킬 수 있는 현상을 뜻한다. 염색체의후성유전학적 상태는 히스톤 변형, DNA 변형 그리고 RNAi에 의한 유전자 침묵 등에 의해 조절된다. 본 총설에서는 배아줄기세포에서의 후성 유전학적 조절에 영향을 주는 요인으로서 히스톤(histone)의 메틸화에 초점을 맞추었다. 배아줄기세포에서 발현되는 유전자의 조절에는 두 가지 단백질 복합체가 관여한다. Polycomb repressive complex 2(PRC2)는 EED,EZH2, SUZ1를 주요인자로 포함하며, H3K27의 trimethylation(H3K27me3)을 증가시킴으로써 유전자의 발현을 억제한다. 이와는 대조적으로 Trithorax group(TrxG) 복합체는 주요인자로 MLL family를 포함하며, H3K4의 trimethylation(H3K4me3)시킴으로써 유전자의 발현을 활성화한다. PRC2 및 TrxG는 다양한 보조 단백질을 포함한다. 배아줄기세포에서 후성유전학적 조절의 두드러진 특징은 H3K27me3과 H3K4me3이 동시에 나타나는 이가 상태(bivalent state)이다. PRC2와 TrxG 복합체 그리고 H3K4나 K3K27의 메틸화에 특이적으로 작용하는 탈메틸효소(demethylase)가 한데 어우러져 배아줄기세포에서 만능성 관련 유전자와 발달 관련 유전자의 발현을 조절함으로써 줄기세포의 유지 및 분화에 기여한다. 따라서 후성유전학적 조절인자들에 대한 보다 자세한 연구는 배아줄기세포를 보다 잘 이해하고 활용하는데 도움을 줄 것이다
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석현정,김영은,박정아,이영희 한국발생생물학회 2011 발생과 생식 Vol.15 No.1
Embryonic stem (ES) cells can self-renew maintaining the undifferentiated state. Self renewal requires many factors such as Oct4, Sox2, FoxD3, and Nanog. NF-κB is a transcription factor involved in many biological activities. Expression and activity of NF-kB increase upon differentiation of ES cells. Reportedly, Nanog protein directly binds to NF-κB protein and inhibits its activity in ES cells. Here, we found a potential binding site of NF-κB in the human Nanog promoter and postulated that NF-κB protein may regulate expression of the Nanog gene. We used human embryonic carcinoma (EC) cells as a model system of ES cells and confirmed decrease of Nanog and increase of NF-κB upon differentiation induced by retinoic acid. Although deletion analysis on the DNA fragment including NF-κB binding site suggested involvement of NF-κB in the negative regulation of the promoter, site-directed mutation of NF-κB binding site had no effect on the Nanog promoter activity. Furthermore, no direct association of NF-κB with the Nanog promoter was detected during differentiation. Therefore, we conclude that NF-κB protein may not be involved in transcriptional regulation of Nanog gene expression in EC cells and possibly in ES cells.
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