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Asian-Australas J Anim Sci > Accepted Articles
DOI: https://doi.org/10.5713/ajas.19.0654    [Accepted] Published online October 22, 2019.
Histone deacetylases inhibitor and RAD51 increase TALEN-mediated homologous recombination on the bovine β-casein gene locus
Da Som Park1  , Se Eun Kim1  , Deog-Bon Koo2  , Man-Jong Kang1,* 
1Department of Animal Science, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 61186, Korea
2Department of Biotechnology, College of Engineering, Daegu University, 201 Daegudae-ro, Jillyang, Gyeongsan, Gyeongbuk 38453, Korea
Correspondence:  Man-Jong Kang, Tel: +82-62-530-2113, Fax: +82-62-530-2129, Email: mjkang@chonnam.ac.kr
Received: 13 August 2019   • Revised: 29 August 2019   • Accepted: 14 October 2019
Abstract
Objective
The efficiency of the knock-in process is very important to successful gene editing in domestic animals. Recently, it was reported that transient loosening of the nucleosomal folding of transcriptionally inactive chromatin might have the potential to enhance homologous recombination efficiency. The objective of this study was to determine whether histone deacetylases (HDAC) inhibitor and RAD51 expression were associated with increased knock-in efficiency on the β-casein gene locus in mammary alveolar-large T antigen (MAC-T) cells using the transcription activator-like effector nucleases (TALEN) system. Method: MAC-T cells were treated with HDAC inhibitors, valproic acid, trichostatin A, or sodium butyrate for 24 h, then transfected with a knock-in vector, RDA51 expression vector and TALEN to target the β-casein gene. After 3 days of transfection, the knock-in efficiency was confirmed by PCR and DNA sequencing of the target site.
Results
The level of HDAC2 protein in MAC-T cells was decreased by treatment with HDAC inhibitors. The knock-in efficiency in MAC-T cells treated with HDAC inhibitors was higher than in cells not treated inhibitors. However, the length of the homologous arm of the knock-in vector made no difference in the knock-in efficiency. Furthermore, DNA sequencing confirmed that the precision of the knock-in was more efficient in MAC-T cells treated with sodium butyrate.
Conclusion
These results indicate that chromatin modification by HDAC inhibition and RAD51 expression enhanced the homologous recombination efficiency on the β-casein gene locus in MAC-T cells.
Keywords: Knock-in; HDAC Inhibitor; Homologous Recombination; RAD51; TALEN
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