The widely used Ucp1-CreEvdr transgene elicits complex developmental and metabolic phenotypes.

Bacterial artificial chromosome transgenic models, including most Cre-recombinases, enable potent interrogation of gene function in vivo but require rigorous validation as limitations emerge. Due to its high relevance to metabolic studies, we performed comprehensive analysis of the Ucp1-CreEvdr line which is widely used for brown fat research. Hemizygotes exhibited major brown and white fat transcriptomic dysregulation, indicating potential altered tissue function. Ucp1-CreEvdrhomozygotes also show high mortality, growth defects, and craniofacial abnormalities. Mapping the transgene insertion site revealed insertion in chromosome 1 accompanied by large genomic alterations disrupting several genes expressed in a range of tissues. Notably, Ucp1-CreEvdr transgene retains an extra Ucp1 gene copy that may be highly expressed under high thermogenic burden. Our multi-faceted analysis highlights a complex phenotype arising from the presence of the Ucp1-CreEvdr transgene independently of the intended genetic manipulations. Overall, comprehensive validation of transgenic mice is imperative to maximize discovery while mitigating unexpected, off-target effects. HighlightsO_LIHemizygous Ucp1-CreEvdr mice exhibit substantial brown and white fat tissue dysregulation. C_LIO_LIHomozygous Ucp1-CreEvdr mice display high mortality, growth defects, and craniofacial abnormalities. C_LIO_LIThe Ucp1-CreEvdr transgene integration resulted in major genomic disruptions affecting multiple genes. C_LIO_LIThe Ucp1-CreEvdr transgene retains a possibly functional extra Ucp1 copy. C_LI