The decrease in Rad51 and DNA ligase IV nuclear protein expression in Msh2 knockdown HC11 cells induced the low CRISPR/Cas9-mediated knock-in efficiency at the β-casein gene locus
| Title: | The decrease in Rad51 and DNA ligase IV nuclear protein expression in Msh2 knockdown HC11 cells induced the low CRISPR/Cas9-mediated knock-in efficiency at the β-casein gene locus |
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| Authors: | Ga-Yeon Kim, Man-Jong Kang |
| Source: | Animal Bioscience, Vol 38, Iss 3, Pp 560-567 (2025) |
| Publisher Information: | Asian-Australasian Association of Animal Production Societies, 2025. |
| Publication Year: | 2025 |
| Collection: | LCC:Zoology |
| Subject Terms: | clustered regularly interspaced short palindromic repeats/crispr-associated 9 (crispr/cas9), dna mismatch repair (mmr), homologous recombination (hr), non-homologous end joining (nhej), Zoology, QL1-991 |
| More Details: | Objective Successful gene editing technology is crucial in molecular biology and related fields. An essential part of an efficient knock-in system is increasing homologous recombination (HR) efficiency in the double-strand break (DSB) repair pathways. Interestingly, HR is closely related to the DNA mismatch repair (MMR) pathway, whereby MMR-related gene Msh2 recognizes a mismatch of nucleotides in recombinant intermediates or gene conversion formed during HR. This study aimed to investigate how the knockdown of Msh2 affects HR-mediated knock-in efficiency at the mouse β-casein locus. Therefore, we investigated the effect of inhibiting Msh2 expression on the expression of the HR-related gene Rad51 and the key enzyme DNA ligase IV involved in non-homologous end joining (NHEJ). Methods The knock-in vector targeting the mouse β-casein gene locus, programmed guide RNA, and Msh2 siRNA expression vector were co-transfected in HC11 cells, or only the Msh2 siRNA expression vector was transfected. Knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA and protein expression of Msh2, HR-related gene Rad51, and NHEJ-related gene DNA ligase IV were evaluated by quantitative reverse transcription PCR (RT-qPCR) and Western blot analysis. Results The knock-in vector efficiency at the mouse β-casein gene locus significantly decreased upon Msh2 knockdown in HC11 mouse mammary epithelial cells (HC11 cell). Additionally, the knockdown of the DNA MMR-related gene Msh2 protein significantly downregulated the nuclear protein expression of the HR-related Rad51 and NHEJ-related DNA ligase IV genes. Conclusion The decreased Msh2 protein expression in the nucleus downregulated the Rad51 and ligase IV protein expressions. Consequently, reduced Rad51 expression results in decreased knock-in efficiency. |
| Document Type: | article |
| File Description: | electronic resource |
| Language: | English |
| ISSN: | 2765-0189 2765-0235 |
| Relation: | http://www.animbiosci.org/upload/pdf/ab-24-0206.pdf; https://doaj.org/toc/2765-0189; https://doaj.org/toc/2765-0235 |
| DOI: | 10.5713/ab.24.0206 |
| Access URL: | https://doaj.org/article/2b42153382554a02bd569772edc126f3 |
| Accession Number: | edsdoj.2b42153382554a02bd569772edc126f3 |
| Database: | Directory of Open Access Journals |
| ISSN: | 27650189 27650235 |
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| DOI: | 10.5713/ab.24.0206 |
| Published in: | Animal Bioscience |
| Language: | English |