Earticle

초록

영어

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.

목차

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Construction of knock-in vector
Cell culture and transfection
Reverse transcription followed by quantitative polymerase chain reaction
Western blot analysis
Analysis of knock-in efficiency by polymerase chain reaction
Statistical analysis
RESULTS
Construction of the knock-in vector for hEPO gene expression in the mouse beta-casein locus
The mRNA and protein expression levels of DNA MMR-related genes, Rad51, and DNA Ligase IV in HC11 cells treated with CdCl2
The CRISPR/Cas9-mediated knock-in efficiency in HC11 cells treated with a high-dose of CdCl2
DISCUSSION
CONCLUSION
REFERENCES

키워드

저자

• Ga-Yeon Kim [ Department of Animal Science, College of Agriculture and Life Science, Chonnam National University, Gwangju 61186, Korea ]
• Man-Jong Kang [ Department of Animal Science, College of Agriculture and Life Science, Chonnam National University, Gwangju 61186, Korea ] Corresponding Author

참고문헌

발행기관

간행물

관심저널 등록

• 간행물명

  Journal of Animal Reproduction and Biotechnology

• 간기

  계간

• pISSN

  2671-4639

• eISSN

  2671-4663

• 수록기간

  2019~2025

• 십진분류

  KDC 527 DDC 636