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