CRISPR/Cas9-Mediated Knockout Reduces Triacylglycerol Content and Key Milk Fat Metabolism Gene Expression in MAC-T Cells.

TARDBP mediates milk fat secretion in mice by binding to UG-rich sequences in the 3' untranslated region (UTR) of and mRNA and enhancing their mRNA stability. However, its role in bovine milk lipid metabolism remains unclear. To investigate this, we generated knockout (KO) MAC-T cells using CRISPR/Cas9 technology, quantified triacylglycerol (TAG) levels in both cells and culture supernatant, and examined the impact of on mRNA levels in MAC-T cells through transcriptome sequencing. We found that deletion of reduced TAG content in both MAC-T cells and the supernatant, as well as decreased mRNA levels of , , , , and . However, the expression of and was unaffected in bovine MAC-T cells. Sequence analysis further revealed TG-rich sequences within bovine and but not in , , , or and . These findings suggest that may regulate bovine lipid metabolism through a mechanism distinct from that described in mice. This study provides new insights into the molecular role of in bovine milk fat metabolism and establishes a foundation for understanding its contribution to dairy cattle breeding and milk quality improvement.