The widely used transgene elicits complex developmental and metabolic phenotypes.

Bacterial artificial chromosome transgenic models, including most , enable potent interrogation of gene function but require rigorous validation as limitations emerge. Due to its high relevance to metabolic studies, we performed comprehensive analysis of the line which is widely used for brown fat research. Hemizygotes exhibited major brown and white fat transcriptomic dysregulation, indicating potential altered tissue function. homozygotes 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, transgene retains an extra 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 transgene independently of the intended genetic manipulations. Overall, comprehensive validation of transgenic mice is imperative to maximize discovery while mitigating unexpected, off-target effects.