** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** COW MILK IS A MAJOR SOURCE OF NUTRIENTS FOR HUMANS. A HOLSTEIN COW ON AVERAGE PRODUCES 35 KG OF MILK THAT CONTAINS 1 KG PROTEIN, 1.3 KG FAT, AND 1.6 KG LACTOSE PER DAY DURING LACTATION. UNDERLYING THIS EXTRAORDINARY MILK-PRODUCING ABILITY OF THE BOVINE MAMMARY GLAND ARE COPIOUS MILK-PRODUCING EPITHELIAL CELLS AND ROBUST GENE TRANSCRIPTION IN THESE CELLS. SURPRISINGLY, LITTLE IS KNOWN ABOUT THE MECHANISMS THAT MEDIATE THESE CHANGES. THEREFORE, THE OVERALL GOAL OF THIS PROJECT IS TO UNDERSTAND THE MECHANISMS THAT MEDIATE GENE EXPRESSION AND THEREBY MILK PRODUCTION IN THE MAMMARY GLAND OF DAIRY COWS. BASED ON OUR PREVIOUS AND PRELIMINARY FINDINGS, WE HYPOTHESIZE THAT TRANSCRIPTION FACTORS INCLUDING STAT5 PLAY IMPORTANT ROLES IN REGULATING GENE EXPRESSION IN THE MAMMARY GLAND OF LACTATING COWS. WE WILL TEST THIS HYPOTHESIS BY DETERMINING (1) IF STAT5 AND OTHER SELECTED TRANSCRIPTION FACTORS BIND TO GENES INVOLVED IN MILK PRODUCTION IN THE MAMMARY GLAND OF COWS, (2) THE EFFECTS OF KNOCKDOWN AND OVEREXPRESSION OF STAT5 AND OTHER SELECTED TRANSCRIPTION FACTORS ON MILK PRODUCTION PROCESSES IN MAMMARY EPITHELIAL CELL MODELS, AND (3) THE FUNCTIONAL IMPORTANCE OF SELECTED DNA REGIONS BOUND BY STAT5 TO THE EXPRESSION OF MILK CASEIN PROTEIN GENES. WE WILL CONDUCT THESE STUDIES IN BOTH ANIMALS AND CELL CULTURE USING A VARIETY OF TECHNIQUES INCLUDING THE CRISPR/CAS9 SYSTEM. THESE STUDIES WILL GENERATE NEW INSIGHTS INTO HOW GENE EXPRESSION IN THE LACTATING MAMMARY GLAND IS REGULATED, AND THESE NEW INSIGHTS COULD BE USED TO DEVELOP NOVEL STRATEGIES TO IMPROVE MILK PRODUCTION OR PRODUCTION EFFICIENCY, OR, TO MANAGE MILK COMPOSITION IN COWS.