• 한국동물생명공학회지
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• 제38권3호
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• pp.99-108
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• 2023

Background: Efficient gene editing technology is needed for successful knock-in. Homologous recombination (HR) is a major double-strand break repair pathway that can be utilized for accurately inserting foreign genes into the genome. HR occurs during the S/G2 phase, and the DNA mismatch repair (MMR) pathway is inextricably linked to HR to maintain HR fidelity. This study was conducted to investigate the effect of inhibiting MMR-related genes using CdCl₂, an MMR-related gene inhibitor, on HR efficiency in HC11 cells. Methods: The mRNA and protein expression levels of MMR-related genes (Msh2, Msh3, Msh6, Mlh1, Pms2), the HR-related gene Rad51, and the NHEJ-related gene DNA Ligase IV were assessed in HC11 cells treated with 10 μM of CdCl₂ for 48 hours. In addition, HC11 cells were transfected with a CRISPR/sgRNA expression vector and a knock-in vector targeting Exon3 of the mouse-beta casein locus, and treated with 10 μM cadmium for 48 hours. The knock-in efficiency was monitored through PCR. Results: The treatment of HC11 cells with a high-dose of CdCl₂ decreased the mRNA expression of the HR-related gene Rad51 in HC11 cells. In addition, the inhibition of MMR-related genes through CdCl₂ treatment did not lead to an increase in knock-in efficiency. Conclusions: The inhibition of MMR-related gene expression through high-dose CdCl₂ treatment reduces the expression of the HR-related gene Rad51, which is active during recombination. Therefore, it was determined that CdCl₂ is an inappropriate compound for improving HR efficiency.

• Asian Pacific Journal of Cancer Prevention
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• 제16권18호
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• pp.8553-8557
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• 2016

Background: Accurate dose assessment and correct identification of irradiated from non-irradiated people are goals of biological dosimetry in radiation accidents. Objectives: Changes in the FDXR and the RAD51 gene expression (GE) levels were here analyzed in response to total body exposure (TBE) to a 6 MV x-ray beam in rats. We determined the accuracy for absolute quantification of GE to predict the dose at 24 hours. Materials and Methods: For this in vivo experimental study, using simple randomized sampling, peripheral blood samples were collected from a total of 20 Wistar rats at 24 hours following exposure of total body to 6 MV X-ray beam energy with doses (0.2, 0.5, 2 and 4 Gy) for TBE in Linac Varian 2100C/D (Varian, USA) in Golestan Hospital, in Ahvaz, Iran. Also, 9 rats was irradiated with a 6MV X-ray beam at doses of 1, 2, 3 Gy in 6MV energy as a validation group. A sham group was also included. After RNA extraction and DNA synthesis, GE changes were measured by the QRT-PCR technique and an absolute quantification strategy by taqman methodology in peripheral blood from rats. ROC analysis was used to distinguish irradiated from non-irradiated samples (qualitative dose assessment) at a dose of 2 Gy. Results: The best fits for mean of responses were polynomial equations with a R2 of 0.98 and 0.90 (for FDXR and RAD51 dose response curves, respectively). Dose response of the FDXR gene produced a better mean dose estimation of irradiated "validation" samples compared to the RAD51 gene at doses of 1, 2 and 3 Gy. FDXR gene expression separated the irradiated rats from controls with a sensitivity, specificity and accuracy of 87.5%, 83.5% and 81.3%, respectively, 24 hours after dose of 2 Gy. These values were significantly (p<0.05) higher than the 75%, 75% and 75%, respectively, obtained using gene expression of RAD51 analysis at a dose of 2 Gy. Conclusions: Collectively, these data suggest that absolute quantification by gel purified quantitative RT-PCR can be used to measure the mRNA copies for GE biodosimetry studies at comparable accuracy to similar methods. In the case of TBE with 6MV energy, FDXR gene expression analysis is more precise than that with RAD51 for quantitative and qualitative dose assessment.

• Journal of Microbiology
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• 제41권1호
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• pp.46-51
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• 2003

RAD6 in yeast mediates postreplication DNA repair and is responsible for DNA-damage induced mutations. RAD6 encodes ubiquitin-conjugating enzyme that is well conserved among eukaryotic organisms. However, the molecular targets and consequences of their ubiquitination by Rad6 have remained elusive. In Aspergillus nidulans, a RAD6 homolog has been isolated and shown to be an allele of uvs). We screened a CDNA library to isolate UVSJ-interacting proteins by the yeast two-hybrid system. JIPA was identified as an interactor of UVSJ. Their interaction was confirmed in vitro by a GST-pull down assay. JIPA was also able to interact with mutant UVSJ proteins, UVSJl and the active site cysteine mutant UVSJ-C88A. The N- and the C-terminal regions of UVSJ required for the interaction with UVSH, a RAD18 homolog of yeast which physically interacts with Rad6, were not necessary for the JIPA and UVSJ interactions. About 1.4 kb jipA transcript was detected in Northern analysis and its amount was not significantly increased in response to DNA-damaging agents. A genomic DNA clone of the jipA gene was isolated from a chromosome I specific genomic library by PCR-sib selection. Sequence determination of genomic and cDNA of jipA revealed an ORF of 893 bp interrupted by 2 introns, encoding a putative polypeptide of 262 amino acids. JIPA has 33% amino acid sequence identity to TIP41 of Saccharomyces cerevisiae which negatively regulates the TOR signaling pathway.

• 한국생태학회:학술대회논문집
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• 한국생태학회 1999년도 국제 Symposium 및 '99 춘계 공동 학술 발표대회
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• pp.133.1-133.1
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• 1999

• Journal of the Korean Academy of Child and Adolescent Psychiatry
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• 제8권2호
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• pp.207-216
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• 1997

본 연구는 반응성 애착장애(reactive attachment disorder：향후 RAD라 칭함)집단과 발달성 언어장애(developmental language disorder：향후 DLD라 칭함)집단을 대상으로 외관상 유사한 사회성 문제를 지니고 있으나, 타인과의 사회적 접촉의 시도라는 좀 더 근본적인 의사소통의 의도에서는 집단간에 어떤 차이를 보이는지 알아보기 위해 생활 연령과 언어 연령으로 두 집단을 짝지어 의사소통 의도의 빈도와 주로 사용하는 의사소통 의도의 유형 및 발달 단계를 비교하였다. 그 결과는 DLD아동의 의도 표현 방법이 RAD아동보다 세련되고 정교화 되었음을 보여 주었으며, DLD아동이 RAD아동보다 의사소통하려는 의도를 더 많이 가지고 있음을 나타냈다. 그리고 두집단이 사용하는 의사소통 의도의 내용을 살펴보았더니, DLD아동은 사회적 상호작용>공유적 주의>행동 통제의 순으로, RAD아동은 행동 통제>사회적 상호작용>공유적 주의의 순으로 의사소통하려는 의도를 나타냄으로써, 주로 사용하는 의도의 내용 범주간에도 두 집단간에 차이를 보였다. 또한, 사용한 의사소통 의도의 다양성 비교에서도 총 12가지의 의사소통 의도에서 DLD아동이 RAD아동보다 더 다양한 수의 의사소통 의도를 나타냈다. 이들 결과를 종합해 볼 때, DLD아동보다는 RAD아동에게서 의사소통의도로 잰 사회적 결함의 심각성이 더 드러났음을 알 수 있다.

• 대한기관식도과학회:학술대회논문집
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• 대한기관식도과학회 1987년도 제21차 학술대회 연제순서 및 초록
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• pp.22.2-22
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• 1987

경동맥 파열은 악성 종양으로 인한 두경부 수술의 1-5%에서 발생하고 80%의 사망률과 생존자의 50%에서 신경학적 후유증을 나타낸다. 저자들은 수술전ㆍ후 방사선치료를 받은 환자 2례에서 경동맥파열을 경험하였다. 제 1례는 51세의 남자로서 후두암($T_4$NoMx)으로 후두전적출술과 6120 rad의 수술후 방사선 치료 후 좌측 악하 부위에 악성 종양의 경동맥 침윤으로 시험적 수술후 6일만에 경동맥 파열로 사망하였다. 제 2례는 51세의 남자로서 하인두암($T_3$$N_2$Mx)으로 7200 rad의 수술전 방사선치료후 악성 종양의 재발로 후두전적출술과 일측의 근치적 경부곽청술후 누공을 형성하여 수술후 14일만에 경동맥 파열로 사망하였다.

• Molecules and Cells
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• 제39권7호
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• pp.550-556
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• 2016

During meiosis, exchange of DNA segments occurs between paired homologous chromosomes in order to produce recombinant chromosomes, helping to increase genetic diversity within a species. This genetic exchange process is tightly controlled by the eukaryotic RecA homologs Rad51 and Dmc1, which are involved in strand exchange of meiotic recombination, with Rad51 participating specifically in mitotic recombination. Meiotic recombination requires an interaction between homologous chromosomes to repair programmed double-strand breaks (DSBs). In this study, we investigated the budding yeast meiosis-specific proteins Hop2 and Sae3, which function in the Dmc1-dependent pathway. This pathway mediates the homology searching and strand invasion processes. Mek1 kinase participates in switching meiotic recombination from sister bias to homolog bias after DSB formation. In the absence of Hop2 and Sae3, DSBs were produced normally, but showed defects in the DSB-to-single-end invasion transition mediated by Dmc1 and auxiliary factors, and mutant strains failed to complete proper chromosome segregation. However, in the absence of Mek1 kinase activity, Rad51-dependent recombination progressed via sister bias in the $hop2{\Delta}$ or $sae3{\Delta}$ mutants, even in the presence of Dmc1. Thus, Hop2 and Sae3 actively modulate Dmc1-dependent recombination, effectively progressing homolog bias, a process requiring Mek1 kinase activation.

• Animal cells and systems
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• 제3권4호
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• pp.413-415
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• 1999

Our previous report demonstrated that the rhp51$^＋$, a recA and RAD51 homolog of the fission yeast, encodes three transcripts of 1.9, 1.6 and 1.3 kb which have at least six polyadenylation sites. The 3'-end of the gene alone can direct the formation of multiple, discrete 3'ends of the transcripts. To identify the regulatory element required for the 3'-end formation of -rhp51$^＋$ deletion mapping analysis was performed. Northern blot analysis revealed that the 254-bp DNA fragment including 4 distinct poly (A) sites downstream from the Hindlll site, is crucial for normal 3'-end formation. Deletion of the 3'-terminal AU rich region caused appearance of read-through RNA, leading to enhancement of survival rate of the rhp51 deletion mutant in response to DNA damaging agent, methylmethane sulfonate (MMS). The results imply that the rhp51$^＋$ system may be useful for molecular analysis of the 3'-end formation of RNA in the fission yeast.

• 한국동물학회:학술대회논문집
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• 한국동물학회 2001년도 한국생물과학협회 학술발표대회
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• pp.239.3-240
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• 2001

No Abstract, See Full Text

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.764-764
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• 2005