• BMB Reports
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• 제52권8호
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• pp.482-489
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• 2019

Reproductive biotechnology has developed rapidly and is now able to overcome many birth difficulties due to infertility or the transmission of genetic diseases. Here we introduce the next generation of assisted reproductive technologies (ART), such as mitochondrial replacement technique (MRT) or genetic correction in eggs with micromanipulation. Further, we suggest that the transmission of genetic information from somatic cells to subsequent generations without gametes should be useful for people who suffer from infertility or genetic diseases. Pluripotent stem cells (PSCs) can be converted into germ cells such as sperm or oocytes in the laboratory. Notably, germ cells derived from nuclear transfer embryonic stem cells (NT-ESCs) or induced pluripotent stem cells (iPSCs) inherit the full parental genome. The most important issue in this technique is the generation of a haploid chromosome from diploid somatic cells. We hereby examine current science and limitations underpinning these important developments and provide recommendations for moving forward.

• 식물조직배양학회지
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• 제27권3호
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• pp.227-232
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• 2000

당근 유식물체의 자엽을 절편으로 유도된 배형성능 세포를 여러가지 농도의 카드뮴 (0, 50. 100, 200. 500, 1000 $\mu$M)이 첨가된 MS 액체배지에 배양한 다음 MS 기본배지에 옮겨서 체세포배 발생을 유도했고 한편 카드뮴 처리구와 카드뮴이 처리되지 않은 대조구에서 체세포배를 얻었다. 현탁배양에서 정상적인 2개의 자엽을 갖는 체세포배 발생은 카드뮴 첨가 배지에서 감소하였으나 비정상적인 3개의 자엽을 갖는 체세포배가 많이 관찰되었다. 또한 비정상적인 1개, 3개 및 4개의 자엽을 갖는 체세포배보다 정상적인 2개의 자엽을 갖는 체세포배의 식물체 재생률이 가장 높았다. 카드뮴 처리구에서 발생된 체세포배의 발아율은 자엽수에 관계없이 대조구에서 발생된 체세포배의 발아율에 비해 감소하였다.

• IMMUNE NETWORK
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• 제7권3호
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• pp.117-123
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• 2007

Background: This study was designed to investigate the role of the hepatocyte growth factor (HGF) with regards to differentiation of somatic stem cells originating from the human umbilical cord blood (UCB) into hepatic lineage cells in vitro culture system. Methods: Mononuclear cells from UCB were cultured with and without HGF based on the fibroblast growth factor (FGF)-1, FGF-2, and stem cell factor. The cultured cells were confirmed by immunofluorescent staining analysis with albumin (ALB), cytokeratin-19 (CK-19), and proliferating cell nuclear antigen (PCNA) MoAb. ALB and CK-18 mRNA were also evaluated by reverse transcription-polymerase chain reaction. In order to observe changes in proliferating capacity with respect to the cultured period, CFSE with affinity to proliferating cells were tagged and later underwent flow cytometry. Results: In the HGF-treated group, cultured cells had a large oval shaped appearance with adherent, but easily detachable characteristics. In the HGF-non treated group, these cells were spindle-shaped with strong adherent characteristics. Expressions of ALB and CK-19 were evident in HGF-treated group compared to non-expression of those in to HGF-non treated group. Dual immunostaining analysis of the ALB producing cells showed presence of PCNA in their nuclei, and ALB and CK-18 mRNA were detected on the 21st day of cultured cells in the HGF-treated group. Conclusion: Our findings suggest that HGF has a pivotal role in differentiating somatic stem cells of human UCB into hepatic lineage cells in vitro.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.69-69
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• 2001

The cell cycle phase in which donor nuclei exist prior to nuclear transfer is an important factor governing developmental rates of reconstituted embryos. It was suggested that quiescent G0 and cycling G1 cells could support normal development of reconstituted embryos. In a quest of optimized donor nuclei treatment prior to nuclear transfer, this study was undertaken to examine the cell cycle characteristics of bovine fetal and adult somatic cells when cultured under a variety of culture treatments and the cell cycle change with the lapse of time after trypsinization. This was archived by measuring the DNA content of cells using flow cytometry, Cultured fetal fibroblast cells, adult skin and muscle cells, and cumulus cells were divided by 3 culture treatments; 1) grown to 60-70% confluency (cycling), 2) serum starved culture, 3) culture to confluency. Trypsinized cells were fixed by 70% ethanol and stained with propidium iodide. For one experiment, trypsinized cells were resuspended in DMEM＋10% FBS and incubated for 1.5, 3 and 6 h with occasional shaking before ethanol fixation. Cell cycle phases were determined by flow cytometry enabling calculation of percentages of G0＋G1, S and G2＋M. The majority of cells were in G0＋Gl stage regardless of origin of cells. Cultures that were serum starved or cultured to confluency contained significantly (P<0.05) higher percentages of cells in G0＋G1 (89.5-95.4%). For every cell lines and culture treatments, percentages of cells in existing in G0＋G1 increased with decreasing of the cell size from large to small. In the serum starved and confluency groups, about 98% of small cells were in G0＋G1 Serum starved culture contained higher percentages of small-sized cells (38.5-66.9%) than cycling and confluent cultures regardless of cell lines (P<0.05). After trypsinization of fetal fibroblast and adult skin cells that were serum starved and cultured to confluency, the percentages of cells in G0＋G1 significantly increased by incubation for 1.5(95.7-99.5%) and 3.0 h (95.9-98.6%). The results suggest that the efficient synchronization of bovine somatic cells in G0＋G1 for nuclear transfer can be established by incubation for a limited time period after trypsinization of serum starved or confluent cells.

• Asian-Australasian Journal of Animal Sciences
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• 제14권5호
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• pp.684-692
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• 2001

To find out the effect of oxytocin on somatic cell count and milk production, 12 primiparous and multiparous Murrah buffaloes were selected, immediately after the parturition, from the Institute's buffalo herd. These were divided into two groups of 6 each. Buffaloes of group I did not receive oxytocin injection (control); whereas, buffaloes of group II were administered oxytocin during early lactation (av. 42.50 days). The oxytocin injection was given in doses of 2.5 IU i.m. before the start of milking, to let down the milk, for a period of 5 days. Samples of milk from individual buffaloes were collected for 5 days before (Period I), during (Period II) and after (Period III) from both the group of buffaloes. Milk samples of A. M. and P. M. milking were composited in proposition to milk yields for analysis of milk constituents. Normal values of somatic cell counts in group I of buffaloes varied from 0.54 to $0.75{\times}10^{5}cells/ml$. Mean cytoplasmic particles and epithelial cells varied from 3.68 to $7.19{\times}10^{5}cells/ml$ and 0.13 to $0.54{\times}10^{5}cells/ml$. On percentage basis the epithelial and the total leucocyte count were 60 and 40. Total leucocyte count, in the study varied from 0.17 to $0.69{\times}10^{5}cells/ml$. The differential cell count of milk indicated presence of lymphocytes (16.50 to $61.16{\times}1000$), neutrophil (0.00 to $2.00{\times}1000$) and monocyte (0.00 to $18.16{\times}1000$). Somatic cell count (p<0.01) and epithelial cells (p<0.05) varied between buffaloes and between periods of study. Total leucocyte counts of milk were also significantly varied between periods (p<0.05). The change in fat, lactose, chloride, EC and NEFA concentrations during different periods of study, were highly significant, indicated diurnal variations in different buffaloes during different days of experiment. Administration of oxytocin resulted in increase in somatic cell counts of milk (p<0.01) due to the increases in total leucocyte count (p<0.01) during the treatment period. The differential cell count indicated that oxytocin administration increased lymphocyte number significantly (p<0.01). However, secretion of neutrophil, monocyte and cytoplasmic particles were not affected by oxytocin. Eosinophil and basophil cell, though present in few samples, remain unaffected by oxytocin administration. There was no effect of oxytocin on milk production, composition, pH, EC and NEFA concentration.

• 한국발생생물학회지:발생과생식
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• 제14권2호
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• pp.43-50
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• 2010

최근에 체세포 리프로그래밍 기법을 사용하여 체세포에 몇 가지 전사인자(리프로그래밍 인자)를 넣어줌으로써 유도만능줄기세포(induced pluripotent stem cell, iPS)를 만드는데 성공하였다. 유도만능줄기세포는 배아줄기세포와 유사하게 자가재생 할 수 있는 능력이 있으며, 신체의 모든 타입의 세포로 분화할 수 있는 특징을 가지고 있다. 배아줄기세포와는 달리 면역거부반응이 없다는 점과 윤리적인 문제로부터 자유롭다는 장점이 있어 2006년 Yamanaka 팀이 유도만능줄기세포에 관해 처음 보고한 이후로 이 분야 연구의 급속한 발전이 이루어지고 있다. 하지만 안전성의 문제점 때문에 세포치료제로 사용되기 위해서는 리프로그래밍 인자의 도입 방법 및 새로운 리프로그래밍 인자의 발굴 등 몇 가지 해결해야 할 점들이 남아 있다. 본 종설에서는 유도만능줄기세포를 만드는데 사용된 몇 가지 리프로그래밍 인자에 대해 보고된 연구 내용을 리프로그래밍 인자가 존재하는 세포인 배아줄기세포 및 난자와 배아에서 정리하고자 하며, 리프로그래밍 인자의 연구에 관한 방향에 대해 논의하고자 한다.

• BMB Reports
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• 제51권9호
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• pp.437-443
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• 2018

Non-homologous end joining (NHEJ), and to a lesser extent, the error-free pathway known as homology-directed repair (HDR) are cellular mechanisms for recovery from double-strand DNA breaks (DSB) induced by RNA-guided programmable nuclease CRISPR/Cas. Since NHEJ is equivalent to using a duck tape to stick two pieces of metals together, the outcome of this repair mechanism is prone to error. Any out-of-frame mutations or premature stop codons resulting from NHEJ repair mechanism are extremely handy for loss-of-function studies. Substitution of a mutation on the genome with the correct exogenous repair DNA requires coordination via an error-free HDR, for targeted transgenesis. However, several practical limitations exist in harnessing the potential of HDR to replace a faulty mutation for therapeutic purposes in all cell types and more so in somatic cells. In germ cells after the DSB, copying occurs from the homologous chromosome, which increases the chances of incorporation of exogenous DNA with some degree of homology into the genome compared with somatic cells where copying from the identical sister chromatid is always preferred. This review summarizes several strategies that have been implemented to increase the frequency of HDR with a focus on somatic cells. It also highlights the limitations of this technology in gene therapy and suggests specific solutions to circumvent those barriers.

• 식물조직배양학회지
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• 제22권6호
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• pp.323-327
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• 1995

미나리의 체세포 배발생 과정을 해부학적으로 구명하기 위하여 배발생 기원세포와 캘러스를 광학현미경 및 전자현미경으로 관찰한 결과, 배발생 세포는 hematoxylin에 짙은 보라색으로, 비배발생 세포는 safranin에 적색으로 염색되어 광학현미경하에서 쉽게 구별할 수 있었다. 배발생 캘러스는 많은 수의 원배 및 발육중인 배, 비배발생 세포 등으로 구성되어 있었다. 체세포 배발생은 발육 중인 배나 세포괴의 표피세포에 위치한 배발생 세포의 하나가 분열하거나 세포괴내의 비배발생 세포속에 묻혀 있는 배발생 세포가 분열하여 일어났다. 배발생 과정은 항상 일정한 형태는 아니지만 단세포로부터 일정한 segmentation 과정를 거쳐서 배발생이 진행되는 것으로 나타났다. 투과전자현미경에 의한 관찰에서 배발생 세포는 비배발생 세포에 비하여 세포질이 조밀하고 핵이 대형이며 amyloplast, 인지질체 및 세포소기 관들이 많으며 액포가 없거나 매우 작았다. 이들 세포들은 두터운 세포벽에 의하여 주위의 비배발생 세포와 분리되어 있으며 세포윤곽은 둥글었다. 주사전자현미경으로 관찰한 배발생캘러스는 외부가 그물이 씌워진 형태의 구형의 다양한 크기의 배들과 비교적 크기가 큰 비배발생 세포들이 혼재하였다. 한편 비배발생능 캘러스는 구성세포가 크고 외부에는 gelatin같은 물질로 덮여 있었다.

• Journal of Ginseng Research
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• 제36권4호
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• pp.442-448
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• 2012

Somatic embryogenesis is one of good examples of the basic research for plant embryo development as well as an important technique for plant biotechnology such as medicinally important plants. Single embryos develop into normal plantlets with shoots and roots. Therefore, direct single embryogenesis derived from single cells is highly important for normal plant regeneration. Here we demonstrate that the cyclic secondary somatic embryogenesis in Panax ginseng Meyer is a permanent source of embryogenic material that can be used for genetic manipulations. Secondary somatic embryos were originated directly from the primary somatic embryos on hormone-free Murashige and Skoog medium, and proliferated further in a cyclic manner. EM medium (one third of modified MS medium [MS medium containing half amount of NH4NO3 and KNO3] with 2% to 3% sucrose) favored further development of proliferated secondary somatic embryos into plantlets with root system. The plantlets developed into plants with well-developed taproots in half-strength Schenk and Hildebrandt basal medium supplemented with 0.5% activated charcoal.

• 한국수정란이식학회지
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• 제23권2호
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• pp.67-76
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• 2008

Since the birth of Dolly using fully differentiated somatic cells as a nuclear donor, viable clones were generated successfully in many mammalian species. These achievements in animal cloning demonstrate developmental potential of terminally differentiated somatic cells. At the same time, the somatic cell nuclear transfer (SCNT) technique provides the opportunities to study basic and applied biosciences. However, the efficiency generating viable offsprings by SCNT remains extremely low. There are several explanations why cloned embryos cannot fully develop into viable animals and what factors affect developmental potency of reconstructed embryos by the SCNT technique. The most critical and persuasive explanation for inefficiency in SCNT cloning is incomplete genomic reprogramming, such as DNA methylation and histone modification. Numerous studies on genomic reprogramming demonstrated that incorrect DNA methylation and aberrant epigenetic reprogramming are considerably correlated with abnormal development of SCNT cloned embryos even though its mechanism is not fully understood. The SCNT technique is useful in cloning farm animals because pluripotent stem cells are not established in farm animal species. Therapeutic cloning combined with genetic manipulation will help to control various human diseases. Also, the SCNT technique provides a chance to overcome excessive demand for the organs by production of transgenic animals as xenotransplantation resources. Here, we describe the factors affecting the efficiency of generating cloned farm animals by the SCNT technique and discuss future directions of animal cloning by SCNT to improve the cloning efficiency.