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-CreEvdr homozygotes also show high mortality, tissue specific 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 intended genetic manipulations. Overall design: Experimental mice were generated by breeding UCP1-CreEvdr heterozygous mice. Mice were sacrified and intersacpular brow and posterior subcutaneous adipose tissues obtained at 6 weeks of age without farther manipulation. RNA was isolated using Trizol and RNAeasy colums.细菌人工染色体（bacterial artificial chromosome）转基因模型（涵盖多数Cre重组酶（Cre-recombinase）系统）可实现体内基因功能的高效解析，但随着其局限性逐渐显现，需开展严格的验证工作。鉴于该工具与代谢研究高度相关，我们对广泛应用于棕色脂肪研究的Ucp1-CreEvdr品系开展了全面分析。半合子小鼠表现出显著的棕色与白色脂肪组织转录组失调，提示其组织功能可能发生改变。Ucp1-CreEvdr纯合子小鼠还表现出高死亡率、组织特异性生长缺陷以及颅面畸形。对转基因插入位点进行定位后发现，该转基因插入于1号染色体，同时伴随大片段基因组变异，破坏了多个在多种组织中表达的基因。值得注意的是，Ucp1-CreEvdr转基因携带额外的Ucp1基因拷贝，该拷贝在高产热负荷下可能呈高表达状态。我们的多维度分析表明，Ucp1-CreEvdr转基因的存在会独立于预设的遗传操作之外，引发复杂的表型。实验设计：通过交配UCP1-CreEvdr杂合子小鼠构建实验小鼠模型。在小鼠6周龄时对其实施安乐死，采集其肩胛间棕色脂肪及后侧皮下脂肪组织，未进行额外操作。采用Trizol试剂与RNAeasy吸附柱分离RNA。