Abstract. Biomedical research tremendously benefits from approaches that enable pharmacological control of gene expression in live laboratory animals. However, current techniques have limitations that often imped the design of transformative experimental paradigms. These include suboptimal temporal dynamics and undesired side effects of inducing drugs on animal physiology and behavior. Our preliminary studies demonstrate that these obstacles can be overcome with new approaches permitting acute regulation of nuclear protein function with the antibiotic, trimethoprim (TMP). This innovative strategy takes advantage of destabilizing domains (DD) that mediate instant degradation of synthesized proteins of interest (POI). TMP restores the stability of DD-POIs by binding to DD tags with high affinity. When compared to other chemical-genetic methods, TMP-inducible stabilization has several advantages: (i) TMP is an inexpensive commercially available small molecule that efficiently penetrates peripheral tissues and the blood-brain barrier; (ii) TMP does not produce adverse effects in mammals due to lack of endogenous targets; (iii) DDs can be fused to virtually any protein of interest; and (iv) TMP stabilizes translated proteins with rapid time-course that does not depend on rates of mRNA synthesis. We therefore hypothesize that expression of genetically encoded DD-POIs in model organisms will facilitate a broad spectrum of studies that have not been previously feasible for technical reasons. Here we will: 1) Develop a versatile toolbox for TMP-inducible recombination of chromosomal and episomal DNA with destabilized Cre recombinase (DD-Cre). This method can be used with numerous mouse lines carrying loxP- flanked alleles, and to drive tissue-specific expression of genes of interest with recombinant viruses; 2) Leverage TMP-inducible stabilization of site-specific transcriptional repressors (DD-TR) for acute silencing of multiple genes that act in the same pathway, a task that cannot be performed with other methods. Our qualified interdisciplinary team will rely on mouse models to validate these tools in vivo. We will express DD-Cre and DD-TRs in the brain to systematically characterize their sensitivity to TMP, kinetics of stabilization, and activity on substrates. Moreover, we will exploit these novel systems in proof-of-principle neurobehavioral experiments to investigate the mechanisms of learning and memory.