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.

Increasing the efficiency of HR (homologous recombination) is important for a successful knock-in. Rad51 is mainly involved in homologous recombination and is associated with strand invasion. The HR-related mismatch repair system maintains HR fidelity by heteroduplex rejection and repair. Therefore, the purpose of this study is to control Rad51, which plays a critical role in HR, through UV-induced DNA damage. It is also to confirm the effect on the expression of MMR related genes (Msh2, Msh3, Msh6, Mlh1, Pms2) and HR-related genes closely related to HR through treatment with the MMR inhibitor CdCl2. The mRNA expression of Rad51 gene was confirmed in both HC11 cells and mouse testes, but the mRNA expression of Dmc1 gene was confirmed only in mouse testes. The protein expression of Rad51 and Dmc1 gene increased in UV-irradiated HC11 cells. After 72 hours of treatment with 1 μm of CdCl2, the mRNA expression level of Msh3, Pms2, and Rad51 decreased, but the mRNA expression level of Msh6 and Mlh1 increased in HC11 cells. There was no significant difference in Msh2 mRNA expression between CdCl2 untreated-group and the 72 hours treated group. In conclusion, HR-related gene (Rad51) was increased by UV-induced DNA damage. Treatment of the MMR inhibitor CdCl2 in HC11 cells decreased the mRNA expression of Rad51.

지질과학 분야에서 암석의 생성 시기, 지각과 맨틀 진화연구의 기초자료로 활용되는 Sr 동위원소비는 열이온화 질량분석기(thermal ionization mass spectrometry, TIMS) 혹은 다검출기 유도결합 플라즈마 질량분 석기(multi-collector plasma ionization mass spectrometry, MC-ICP-MS)와 같은 질량분석기를 이용하여 측정할 수 있다. 이 기술보고에서는, Sr 동위원소비 측정시, 원소의 불완전한 화학적 분리가 Sr 동위원소비의 참값 (true value)에 어떤 영향을 미치는지를 비교하였다. 실험에는 상업용 레진, NBS987(NIST SRM987) Sr 동위 원소 표준물질 그리고 일본지질조사소의 암석표준시료 JG1a, JB3, JA1를 이용하였다. 비교실험 결과, NBS987 Sr 동위원소 표준시료, 일본지질조사소의 암석표준시료 JG1a, JB3, JA1 모두 불완전한 분리에 의해 Rb이 남 아있는 경우 87Sr/86Sr의 측정값이 변하는 것이 명확하게 관찰된다. 이는 질량분석기 특히 MC-ICP-MS로 동위 원소비를 측정하고자 하는 경우, 동종동위원소의 간섭에 대한 보정에도 불구하고 측정값은 참값에서 벗어나 므로 완전한 분리가 중요한 인자임을 지시해준다. 그러므로 MC-ICP-MS를 이용한 Sr 동위원소비 측정결과를 보고할 때는, 동종동위원소에 의한 영향을 판단할 수 있도록 Sr의 동위원소 전체의 측정강도와 더불어 85Rb의 측정강도도 함께 보고돼야 할 것이다.

Rad51 is a key component of homologous recombination (HR) to repair DNA double-strand breaks and it forms Rad51 recombinase filaments of broken single-stranded DNA to promote HR. In addition to its role in DNA repair and cell cycle progression, Rad51 contributes to the reprogramming process during the generation of induced pluripotent stem cells. In light of this, we performed reprogramming experiments to examine the effect of co-expression of Rad51 and four reprogramming factors, Oct4, Sox2, Klf4, and c-Myc, on the reprogramming efficiency. Co-expression of Rad51 significantly increased the numbers of alkaline phosphatase-positive colonies and embryonic stem cell-like colonies during the process of reprogramming. Co-expression ofRad51 significantly increased the expression of epithelial markers at an early stage of reprogramming compared with control cells. Phosphorylated histone H2AX (γH2AX), which initiates the DNA double-strand break repair system, was highly accumulated in reprogramming intermediates upon co-expression of Rad51. This study identified a novel role of Rad51 in enhancing the reprogramming efficiency, possibly by facilitating mesenchymal-to-epithelial transition and by regulating a DNA damage repair pathway during the early phase of the reprogramming process.