• BMB Reports
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• 제56권2호
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• pp.102-107
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• 2023

Genome editing using CRISPR-associated technology is widely used to modify the genomes rapidly and efficiently on specific DNA double-strand breaks (DSBs) induced by Cas9 endonuclease. However, despite swift advance in Cas9 engineering, structural basis of Cas9-recognition and cleavage complex remains unclear. Proper assembly of this complex correlates to effective Cas9 activity, leading to high efficacy of genome editing events. Here, we develop a CRISPR/Cas9-RAD51 plasmid constitutively expressing RAD51, which can bind to single-stranded DNA for DSB repair. We show that the efficiency of CRISPR-mediated genome editing can be significantly improved by expressing RAD51, responsible for DSB repair via homologous recombination (HR), in both gene knock-out and knock-in processes. In cells with CRISPR/Cas9-RAD51 plasmid, expression of the target genes (cohesin SMC3 and GAPDH) was reduced by more than 1.9-fold compared to the CRISPR/Cas9 plasmid for knock-out of genes. Furthermore, CRISPR/Cas9-RAD51 enhanced the knock-in efficiency of DsRed donor DNA. Thus, the CRISPR/Cas9-RAD51 system is useful for applications requiring precise and efficient genome edits not accessible to HR-deficient cell genome editing and for developing CRISPR/Cas9-mediated knockout technology.

• Asian-Australasian Journal of Animal Sciences
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• 제29권3호
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• pp.413-418
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• 2016

Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

• 한국약용작물학회지
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• 제18권4호
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• pp.266-272
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• 2010

There is a considerable difference in morphological traits between Bokbunja cultivated in Korea (KCB) and Korea native Rubus coreanus, contrary to the conviction that the cultivated Bokbunja is the domestication of R. coreanus. To infer the phylogenetic relationship of KCB with other Rubus species, we compared the chloroplast DNA spacers of KCB with those of several Rubus species including black raspberry, R. occidentalis. The three chloroplast DNA spacers, atpB~rbcL, trnL~trnF, and trnT~trnL, were amplified using the specific primer pairs and converted to Single Strand Conformational Polymorphism (SSCP) markers. The SSCP makers of the chloroplast DNA spacers showed a considerable variation both within and among Rubus species. In the phylogenetic tree generated by the SSCP markers, KCB accessions were located in the same clade with R. occidentalis, but R. coreanus accessions in the different clade. Also, in the phylogenetic tree by the nucleotide sequences of the chloroplast DNA spacer trnL~trnF, KCB located in the same clade with R. occidentalis but not with R. coreanus. These results suggest that the three KCB accessions share higher similarity with R. occidentalis than with R. coreanus in the three chloroplast DNA spacers.

• Journal of Plant Biotechnology
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• 제31권3호
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• pp.225-230
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• 2004

방사선에 노출된 고추유묘의 잎으로부터 세포핵을 분리하여 단세포전기영동방법인 comet 분석을 통하여 핵 DNA의 손상정도를 조사하였다. Comet 분석에서 꼬리부분의 길이 (T)와 머리부분의 길이 (H)를 측정하여 T/H 비율을 조사하였다. 무처리세포는 T/H 비율이 1.28이었으나 50 및 100 Gy의 방사선을 처리한 세포는 각각 3.54 및 3.39로 방사선처리에 의해 상당량의 핵 DNA가 손상을 입은 것으로 나타났다. Comet의 head-DNA량은 무처리가 76.8%였으나 50 및 100 Gy를 처리한 세포는 각각 55.9% 및 59.5%를 보였다. 고선량의 방사선을 처리하기 전에 미리 20 Gy 이하의 저선량 방사선을 종자에 전처리하였을 경우 종자의 발아 및 생장에 대한 영향은 없었지만, 후속 고선량에 대한 핵 DNA의 손상은 경감되는 경향을 보였다.

• 한국식품과학회지
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• 제31권4호
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• pp.1071-1076
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• 1999

본 연구에서는 김치발효에 관여하는 우세 발효균들의 집락을 분리하고, 발암원인 MNNG에 대하여 이들 분리주들이 갖는 DNA 손상 억제효과를 조사하였다. 이를 위해서 세포의 DNA 손상 및 복구를 측정하는데 이용되는 매우 신속하고 감도가 높은 Single Cell Gel Electrophoresis(SCGE 법, 또는 comet assay)를 사용하였다. 배추김치에서 분리한 3개의 분리주중에서 B-3를 제외한 B-1과 B-2는 양성 대조구와 비교하였을 때 우수한 항유전 독성효과를 나타내었으며(p<0.01), 율무김치의 3 개의 분리주(Y-1, Y-2, Y-3)와 1개의 분리주(Y-4)도 각각 항유전 독성효과를 나타내었다(p<0.05, p<0.01). 총각김치의 5 개 분리주중에서 3개의 분리주와 깍두기의 9개의 분리주중에서 2 개의 분리주들도 각각 항유전 독성효과를 나타내었다(p<0.05, p<0.01).

• Molecular & Cellular Toxicology
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• 제3권3호
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• pp.195-197
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• 2007

Currently praziquantel is used for treatment of not only clonorchiasis but also other trematodes and cestodes. But cure rate of praziquantel is just 60-80% for most trematodes. It needs for the treatment-failed patients to have more drugs. The cause of failure of treatment is not studied. We just know that the blood level of praziquantel is severely different among the people. We guess that this factor may influence the results of treatment. In an endemic area of human clonorchiasis in Heilongjiang Providence, China, 78 subjects were selected for the study. Three doses of 25 mg/kg (total 75 mg/kg) of praziquantel were administered to 78 clonorchiasis patients. After three weeks of treatment, stool examination was undertaken once again to confirm the cured and uncured subjects. To analyze SNP (single nucleotide polymorphism) of CYP3A5 PS2-1, CYP3A5 PS2-2, and CYP3A5*6, PCR method was done with specifically designed primers. The mutation rates of all sites were not significant statistically. The number of subjects was too small, so we need more subjects and other delivery proteins of bile ducts (ex. MRP etc.) were also considered for effects of praziquantel. We analyzed, for the first time, the entire CYP3A5 gene in a French population, using a polymerase chain reaction- single strand conformational polymorphism (PCR-SSCP) strategy.

• Asian-Australasian Journal of Animal Sciences
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• 제18권8호
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• pp.1061-1065
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• 2005

Growth rate is one of the economically important quantitative traits that affect carcass quantity in beef cattle. Two genes, bovine insulin-like growth factor I (IGF-I) and myogenic factor 5 (MYF5), were chosen as candidate genes for growth traits due to their important role in growth and development of mammals. The objectives of this study were to determine gene-specific single nucleotide polymorphism (SNP) markers of the IGF-I and MYF5 positional candidate genes and to investigate their associations with growth traits in Korean cattle. Genotyping of the SNP markers in these candidate genes was carried out using the single strand conformation polymorphism (SSCP) analysis. The frequencies of A and B alleles were 0.72 and 0.28 for IGF-I gene and 0.39 and 0.61 for MYF5 gene, respectively, in Korean cattle population examined. The gene-specific SNP marker association analysis indicated that the SNP genotype in IGF-I gene showed a significant association (p<0.05) with weight at 3 months (W3), and cows with AB genotype had higher W3 than BB genotype cows. The SNP genotype of MYF5 gene was found to have a significant effect (p<0.05) on the weight at 12 months (W12) and average daily gain (ADG), and cows with BB and AB genotypes had higher W12 and ADG compared with cows with AA genotype, respectively. However, no significant association between the SNP genotypes and any other growth traits was detected. The gene-specific SNP markers in the IGF-I and MYF5 candidate genes may be useful for selection on growth traits in Korean cattle.

• Asian-Australasian Journal of Animal Sciences
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• 제28권6호
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• pp.782-787
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• 2015

The myogenic regulatory factors is a family of transcription factors that play a key role in the development of skeletal muscle fibers, which are the main factors to affect the meat taste and texture. In the present study, we performed candidate gene analysis to identify single-nucleotide polymorphisms in the MyoD, Myf5, MyoG, and Mrf4 genes using polymerase chain reaction-single strand conformation polymorphism in 360 Erlang Mountain Chickens from three different housing systems (cage, pen, and free-range). The general linear model procedure was used to estimate the statistical significance of association between combined genotypes and muscle fiber traits of chickens. Two polymorphisms (g.39928301T>G and g.11579368C>T) were detected in the Mrf4 and MyoD gene, respectively. The diameters of thigh and pectoralis muscle fibers were higher in birds with the combined genotypes of GG-TT and TTCT (p<0.05). Moreover, the interaction between housing system and combined genotypes has no significant effect on the traits of muscle fiber (p>0.05). Our findings suggest that the combined genotypes of TT-CT and GG-TT might be advantageous for muscle fiber traits, and could be the potential genetic markers for breeding program in Erlang Mountain Chickens.

• Molecular & Cellular Toxicology
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• 제1권4호
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• pp.230-236
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• 2005

Formaldehyde is a common environmental contaminant found in tobacco smoke, paint, garments, diesel and exhaust, and medical and industrial products. Formaldehyde has been considered to be potentially carcinogenic, making it a subject of major environmental concern. However, only a little information on the mechanism of immunological sensitization and asthma by this compound has been known. So, we performed with Jurkat cell line, a human T lymphocyte, to assess the induction of DNA damage and to identify the DEGs related to immune response or toxicity by formaldehyde. In this study, we investigated the induction of DNA single strand breaks by formaldehyde using single cell gel electrophoresis assay (comet assay). And we compared gene expression between control and formaldehyde treatment to identify genes that are specifically or predominantly expressed by employing annealing control primer (ACP)-based $GeneFishing^{TM}$ method. The cytotoxicity ($IC_{30}$) of formaldehyde was determined above the 0.65 mM in Jurkat cell in 48 h treatment. Based on the $IC_{30}$ value from cytotoxicity test, we performed the comet assay in this concentration. From these results, 0.65 mM of formaldehyde was not revealed significant DNA damages in the absence of S-9 metabolic activation system. And the one differentially expressed gene (DEG) of formaldehyde was identified to zinc finger protein 292 using $GeneFishing^{TM}$ method. Through further investigation, we will identify more meaningful and useful DEGs on formaldehyde, and then can get the information on the associated mechanism and pathway with immune response or other toxicity by formaldehyde exposure.

• 한국산림과학회지
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• 제95권6호
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• pp.631-635
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• 2006

파이토플라스마 증폭 primer, R16F2n/R2를 이용하여 뽕나무 오갈병 파이토플라스마와 대추나무 빗자루병 파이토플라스마에 대하여 SSCP분석법을 이용하여 염기변이 분석을 하였다. 그 결과 뽕나무 및 대추나무 파이토플라스마는 약 1.2 kb PCR 산물을 이용하더라도 뚜렷한 밴드차이를 나타내었다. 유사한 SSCP밴드 패턴을 보이는 두 시료 간의 밴드형태를 뚜렷하게 구별하는 방법을 찾기 위하여 뽕나무 오갈병 파이토플라스마와 대추나무 빗자루병 파이토플라스마의 PCR산물을 혼합한 후 SSCP분석 결과, 전기영동상에서 대추나무 파이토플라스마와 뽕나무 파이토플라스마의 SSCP 밴드패턴 모두를 관찰할 수 있었다. 본 연구 결과, 기존에 약 600bp 크기로 한정된 것으로 알려진 SSCP 분석을 PCR 산물 1.2 kb을 이용하여 유사한 SSCP 밴드패턴을 보이는 두 시료간의 밴드형태를 두 시료의 PCR 산물을 혼합하여 SSCP분석함으로써 뚜렷하게 구별할 수 있었다.