CRISPR/Cas9-Mediated TARDBP 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 <i>BTN1A1</i> and <i>XDH</i> mRNA and enhancing their mRNA stability. However, its role in bovine milk lipid metabolism remains unclear. To investigate this, we generated <i>TARDBP</i> knockout (KO) MAC-T cells using CRISPR/Cas9 technology, quantified triacylglycerol (TAG) levels in both cells and culture supernatant, and examined the impact of <i>TARDBP</i> on mRNA levels in MAC-T cells through transcriptome sequencing. We found that deletion of <i>TARDBP</i> reduced TAG content in both MAC-T cells and the supernatant, as well as decreased mRNA levels of <i>CD36</i>, <i>FABP4</i>, <i>DGAT1</i>, <i>PPARG</i>, and <i>PPARGC1A</i>. However, the expression of <i>BTN1A1</i> and <i>XDH</i> was unaffected in bovine MAC-T cells. Sequence analysis further revealed TG-rich sequences within bovine <i>PPARG</i> and <i>PPARGC1A</i> but not in <i>FABP4</i>, <i>DGAT1</i>, <i>CD36</i>, or <i>BTN1A1</i> and <i>XDH</i>. These findings suggest that <i>TARDBP</i> may regulate bovine lipid metabolism through a mechanism distinct from that described in mice. This study provides new insights into the molecular role of <i>TARDBP</i> in bovine milk fat metabolism and establishes a foundation for understanding its contribution to dairy cattle breeding and milk quality improvement.