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.