• 미생물학회지
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• 제55권3호
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• pp.199-205
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• 2019

Yeast two-hybrid는 특정 단백질에 대한 상호작용 파트너 단백질의 선별을 위한 방법으로 개발되었다. 하지만 대규모 단백질 상호작용체 분석을 수행하기에 요구되는 노동과 대량의 한천배지 사용에 따른 문제에 의해 널리 사용되지 못하고 있다. 따라서 본 연구에서는 새로운 리포터 시스템을 yeast two-hybrid 방법에 도입하여 fluorescence-activated cell sorting (FACS) 또는 magnetic-activated cell sorting (MACS)를 이용하여 상호작용 파트너 단백질을 포함하는 효모 클론을 손쉽게 선별할 수 있도록 하였다. 새로운 리포터 시스템은 c-myc 항원 결정기가 총 10번 반복되는 형태로 효모 표면에 발현되도록 하였으며, p53과 SV40 T항원을 이용한 실험을 통하여 리포터 단백질의 정상적인 발현을 flow cytometry 분석을 통하여 확인하였다. 따라서, 새로운 리포터 시스템을 도입한 yeast two-hybrid 방법은 대규모 상호작용체 분석을 위해 필요한 노력을 현저히 줄일 수 있을 것으로 기대한다.

• 대한족부족관절학회지
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• 제18권3호
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• pp.100-107
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• 2014

Purpose: The purpose of this study is to evaluate the efficacy of mesenchymal stem cell (MSC) isolation by the magnetic-activated cell sorting (MACS) method in tendon tissue-derived cells compared to the colony picking method for isolation of MSCs by picking colony-forming cells. Materials and Methods: Human tendon-derived cells were isolated by enzyme digestion using normal tendon tissues from three donors. We used the magnetic kit and well-known MSC markers (CD90 or CD105) to isolate MSCs in tendon-derived cells using MACS. Cloning cylinders were used to isolate colony-forming cells having MSC characteristics in tendon-derived cells. Colony-forming unit-fibroblast (CFU-F) assay was used to evaluate the self-renewal capacity of cells isolated using the colony picking method or MACS. For comparison of differentiation potentials into osteogenic or adipogenic lineage between two groups, alizarin red S and oil red O staining were performed at 14 days after induction of differentiation in vitro. Results: Flow cytometry results showed that early passage tendon-derived cells expressed CD44 in 99.13%, CD90 in 56.51%, and CD105 in 86.19%. In the CFU-F assay, CD90+ or CD105+ cells isolated with MACS showed larger colony formation in size than cells isolated using the colony picking method. We also observed that CD90+ or CD105+ cells were constantly differentiated into both osteogenic and adipogenic lineages in cells from all donors, whereas cells isolated using the colony picking method were heterogeneous in differentiation potentials to the osteogenic and adipogenic lineages. Conclusion: CD90+ or CD105+ cells isolated using MACS showed superior MSC characteristics in the self-renewal and multi-differentiation capacities compared with cells isolated using the colony picking method.

• 한국축산식품학회지
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• 제40권5호
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• pp.852-859
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• 2020

The muscle stem cells of domestic animals are of interest to researchers in the food and biotechnology industries for the production of cultured meat. For producing cultured meat, it is crucial for muscle stem cells to be efficiently isolated and stably maintained in vitro on a large scale. In the present study, we aimed to optimize the method for the enrichment of pig muscle stem cells using a magnetic-activated cell sorting (MACS) system. Pig muscle stem cells were collected from the biceps femoris muscles of 14 d-old pigs of three breeds [Landrace×Yorkshire×Duroc (LYD), Berkshire, and Korean native pigs] and cultured in skeletal muscle growth medium-2 (SkGM-2) supplemented with epidermal growth factor (EGF), dexamethasone, and a p38 inhibitor (SB203580). Approximately 30% of total cultured cells were nonmyogenic cells in the absence of purification in our system, as determined by immunostaining for cluster of differentiation 56 (CD56) and CD29, which are known markers of muscle stem cells. Interestingly, following MACS isolation using the CD29 antibody, the proportion of CD56⁺/CD29⁺ muscle stem cells was significantly increased (91.5±2.40%), and the proportion of CD56 single-positive nonmyogenic cells was dramatically decreased. Furthermore, we verified that this method worked well for purifying muscle stem cells in the three pig breeds. Accordingly, we found that CD29 is a valuable candidate among the various marker genes for the isolation of pig muscle stem cells and developed a simple sorting method based on a single antibody to this protein.

• Clinical and Experimental Reproductive Medicine
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• 제43권4호
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• pp.199-206
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• 2016

Objective: This study was carried out to investigate the correlations of the sperm DNA fragmentation index (DFI) with semen parameters and apoptosis, and to investigate the effects of density-gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) on reducing the proportion of sperm with DNA fragmentation and protamine deficiency. Methods: Semen analysis and a sperm DNA fragmentation assay were performed to assess the correlations between semen parameters and the DFI in 458 semen samples. Sperm with progressive motility or non-apoptosis were isolated by DGC or MACS, respectively, in 29 normozoospermic semen samples. The effects of DGC or MACS alone and of DGC and MACS combined on reducing the amount of sperm in the sample with DNA fragmentation and protamine deficiency were investigated. Results: The sperm DFI showed a significant correlation (r=-0.347, p< 0.001) with sperm motility and morphology (r=-0.114, p< 0.05) but not with other semen parameters. The DFI ($11.5%{\pm}2.0%$) of semen samples was significantly reduced by DGC ($8.1%{\pm}4.1%$) or MACS alone ($7.4%{\pm}3.9%$) (p< 0.05). The DFI was significantly further reduced by a combination of DGC and MACS ($4.1%{\pm}1.3%$, p< 0.05). Moreover, the combination of DGC and MACS ($1.6%{\pm}1.1%$, p< 0.05) significantly reduced the protamine deficiency rate of semen samples compared to DGC ($4.4%{\pm}3.2%$) or MACS alone ($3.4%{\pm}2.2%$). Conclusion: The combination of DGC and MACS may be an effective method to isolate high-quality sperm with progressive motility, non-apoptosis, high DNA integrity, and low protamine deficiency in clinical use.

• Journal of Microbiology and Biotechnology
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• 제25권4호
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• pp.547-553
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• 2015

The yeast surface-displayed cDNA library has been used to identify unknown antigens. However, when unknown target antigens show moderate-to-low abundance, some modifications are needed in the screening process. In this study, a directional random-primed cDNA library was used to increase the number of candidates for the unknown antigen. To avoid the loss of target yeast clones that express proteins at a low frequency in the cDNA library, a comprehensive monitoring system based on magnetic-activated cell sorting, fluorescence-activated cell sorting, and immunofluorescence was established, and a small number of target yeast cells was successfully enriched. These results showed that our optimized method has potential application for identifying rare unknown antigens of the human monoclonal antibody.

• 한국수정란이식학회지
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• 제33권4호
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• pp.327-336
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• 2018

To date, there are no protocols optimized to the effective separation of spermatogonial stem cells (SSCs) from testicular cells derived from mouse testes, thus hindering studies based on mouse SSCs. In this study, we aimed to determine the most efficient purification method for the isolation of SSCs from mouse testes among previously described techniques. Isolation of SSCs from testicular cells derived from mouse testes was conducted using four different techniques: differential plating (DP), magnetic-activated cell sorting (MACS) post-DP, MACS, and positive and negative selection double MACS. DP was performed for 1, 2, 4, 8, or 16 h, and MACS was performed using EpCAM ($MACS^{EpCAM}$), Thy1 ($MACS^{Thy1}$), or GFR ${\alpha}1$ ($MACS^{GFR{\alpha}1}$) antibodies. The purification efficiency of each method was analyzed by measuring the percentage of cells that stained positively for alkaline phosphatase. DP for 8 h, $MACS^{Thy1}$ post-DP for 8 h, $MACS^{GFR{\alpha}1}$, positive selection double $MACS^{GFR{\alpha}1/EpCAM}$, and negative selection double $MACS^{GFR{\alpha}1/{\alpha}-SMA}$ were identified as the optimal protocols for isolation of SSCs from mouse testicular cells. Comparison of the purification efficiencies of the optimized isolation protocols showed that, numerically, the highest purification efficiency was obtained using $MACS^{GFR{\alpha}1}$. Overall, our results indicate that $MACS^{GFR{\alpha}1}$ is an appropriate purification technique for the isolation of SSCs from mouse testicular cells.

• The Journal of Korean Physical Therapy
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• 제21권3호
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• pp.87-93
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• 2009

Purpose: TThis study examined the effect of repetitive magnetic stimulation (RMS) on the viability and proliferative response of human adipose tissue-derived stromal cells (hATSCs) in vitro. Methods: The hATSCs were cultured primarily from human adipose tissue harvested by liposuction and incubated in a $37^{\circ}C$ plastic chamber. The cells were exposed to a repetitive magnetic field using a customized magnetic stimulator (Biocon-5000, Mcube Technology). The RMS parameters were set as follows: repetition rate=10Hz, 25Hz (stimulus intensity 100%= 0.1 Tesla, at 4cm from the coil), stimulated time= 1, 5, and 20 minutes. Twenty four hours after one application of RMS, the hATSCs were compared with the sham stimulation, which were kept under the same conditions without the application of RMS. The cells were observed by optical microscopy to determine the morphology and assessed by trypan blue staining for cell proliferation. The apoptosis and viability of the hATSCs were also analyzed by fluorescence-activated cell sorting (FACS) analysis of Annexin V and MTT assay. Results: After RMS, the morphology of the hATSCs was not changed and the apoptosis of hATSCs were not increased compared to the sham stimulation. The viability of the cells was similar to the cells given the sham stimulation. Interestingly, the level of hATSC proliferation was significantly higher in all RMS groups. Conclusion: The application of RMS may not cause a change in morphology and viability of hATSCs but can increase the level of cell proliferation in vitro. RMS might be useful as an adjuvant tool in combination with stem cell therapy without adverse effects.

• 한국동물생명공학회지
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• 제36권2호
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• pp.91-98
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• 2021

The objective of this study was to establish a selection process for high quality sperm in bovine semen using sperm separation by magnetic activation (MACS). For this, semen from 21 Nellore bulls was collected using an artificial vagina. To guarantee the presence of pathologies in the ejaculate, animals previously declassified in four consecutive spermiogram were used. Semen was analyzed in five statuses: (1) fresh semen (fresh); (2) density gradient centrifugation (DGC), percoll column; (3) non-apoptotic fraction after separation by MACS (MAC); (4) apoptotic fraction from the separation (MACPOOR); and (5) MAC followed by DGC (MACDGC). Using a computerized analysis system (CASA), motility was measured. The sperm morphology was evaluated by phase contrast, and the supravital test was completed with eosin/nigrosin staining. For DGC, 20 × 10⁶ cells were used in a gradient of 90% and 45% percoll. MACS used 10 × 10⁶ cells with 20 μL of nanoparticles attached to annexin V, and filtered through the MiniMACS magnetic separation column. Membrane integrity was assessed with SYBR-14/IP and mitochondrial potential with JC-1 by flow cytometry. Processing sperm by MACDGC, was more effective in obtaining samples with high quality sperm, verified by the total of abnormalities in the samples: 35.04 ± 2.29%, 21.50 ± 1.47%, 17.30 ± 1.10%, 30.68 ± 1.94% and 10.50 ± 1.46%, respectively for fresh, DGC, MAC, MACPOOR, and MACDGC. The subpopulation of non-apoptotic sperm had a high number of live cells (82.65%), membrane integrity (56.60%) and mitochondrial potential (83.98%) (p < 0.05). These findings suggest that this nanotechnological method, that uses nanoparticles, is efficient in the production of high-quality semen samples for assisted reproduction procedures in cattle.

• Clinical and Experimental Reproductive Medicine
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• 제33권3호
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• pp.171-178
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• 2006

목 적: 인간 배아줄기세포는 재생 의학이나 조직공학에 있어서 큰 잠재적인 능력을 가지고 있는 것으로 알려져 있다. 이들은 다양한 growth factors 처리나 유전자 발현을 변화시켜 특정 세포로 유도 분화 및 분리가 가능하지만 그 효율성은 아직까지 낮은 상태이다. 본 연구에서는 인간 배아줄기세포로부터 비특이적으로 분화된 세포들을 특정 세포 표면 항체를 이용한 magnetic cell sorting (MACS) 방법으로 분리, 배양하여 그들의 특성을 살펴보았다. 연구방법: 미분화 배아줄기세포주(Miz-hESC4)를 물리적인 방법으로 계대 배양하였으며, 부유 배양법으로 배아체 형성을 유도하였다. 배아체의 자발적인 분화를 위해 DMEM에 10% FBS를 첨가하여 2주 동안 배양하였다. 이렇게 분화된 세포들을 CD34, human epithelial antigen (HEA), human fibroblast (HFB)에 대한 항체를 이용한 MACS system으로 각각의 항체에 대한 양성 또는 음성 세포를 분리하였다. 이러한 MACS 분리 세포를 4주 동안 배양하면서 형태적인 변화를 관찰하고 특이 유전자의 발현 양상을 분석하였다. 결 과: 분리 배양한 CD34 양성 세포들은 배양 초기에는 둥근 형태를 나타내다가 배양 후기에는 작은 다각형의 형태로 관찰되었으며, HEA 양성 세포들은 큰 다각형의 형태를 나타내었고, HFB 양성 세포들은 전형적인 방추체 형태로 관찰되었다. 특이 유전자에 대한 RT-PCR 결과에서, CD34 양성 세포들과 HFB양성 세포들에서는 내배엽과 중배엽 관련 유전자의 발현하는 것을 확인할 수 있었고, HEA 양성 세포들에서는 외배엽 관련 유전자인 NESTIN과 NF68KD의 발현을 관찰할 수 있었다. 배양기간이 경과함에 따라 CD34 양성 세포의 특이 유전자 발현 양상이 변화되었다. 결 론: 이상의 결과는 비특이적으로 분화된 인간 배아줄기세포로부터 특이 세포를 MACS 방법을 이용하여 성공적으로 분리할 수 있음을 보여주었다. 따라서, MACS 방법과 특이 세포에 대한 항체는 인간 배아줄기세포의 유도 분화와 특이 세포의 분리에 매우 유용할 것으로 생각된다.

• Asian-Australasian Journal of Animal Sciences
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• 제30권3호
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• pp.439-445
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• 2017

Objective: Production of alpha-1,3-galactosyltransferase (${\alpha}GT$)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous ${\alpha}GT$ knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce ${\alpha}GT$-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Methods: Miniature pig fibroblasts were transfected with ${\alpha}GT$ gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous ${\alpha}GT$ gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-${\alpha}$-1,3-galactose, an epitope produced by ${\alpha}GT$. Using magnetic activated cell sorting, cells with monoallelic disruption of ${\alpha}GT$ were removed. Remaining cells with LOH carrying biallelic disruption of ${\alpha}GT$ were used for the second round NT to produce homozygous ${\alpha}GT$ gene-targeted piglets. Results: Monoallelic mutation of ${\alpha}GT$ gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous ${\alpha}GT$ gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous ${\alpha}GT$ knockout piglets. Conclusion: The present study demonstrates that the time required for the production of ${\alpha}GT$-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a substantial length of time for breeding.