Focal gene amplifications are among the most common cancer-associated mutations¹ but have proven challenging to engineer in primary cells and model organisms. Here we describe a general strategy to engineer large (more than 1 Mbp) focal amplifications mediated by extrachromosomal DNAs (ecDNAs)² in a spatiotemporally controlled manner in cells and in mice. By coupling ecDNA formation with expression of selectable markers, we track the dynamics of ecDNA-containing cells under physiological conditions and in the presence of specific selective pressures. We also apply this approach to generate mice harbouring Cre-inducible Myc- and Mdm2-containing ecDNAs analogous to those occurring in human cancers. We show that the engineered ecDNAs spontaneously accumulate in primary cells derived from these animals, promoting their proliferation, immortalization and transformation. Finally, we demonstrate the ability of Mdm2-containing ecDNAs to promote tumour formation in an autochthonous mouse model of hepatocellular carcinoma. These findings offer insights into the role of ecDNA-mediated gene amplifications in tumorigenesis. We anticipate that this approach will be valuable for investigating further unresolved aspects of ecDNA biology and for developing new preclinical immunocompetent mouse models of human cancers harbouring specific focal gene amplifications.

局灶性基因扩增是最常见的癌症相关突变^{之一 1}，但事实证明，在原代细胞和模式生物中进行工程改造具有挑战性。在这里，我们描述了一种在细胞和小鼠中以时空控制的方式设计由染色体外 DNA （ecDNA^{） 2} 介导的大（超过 1 Mbp）局部扩增的一般策略。通过将 ecDNA 形成与选择标记物的表达相结合，我们跟踪了在生理条件和特定选择压力下含有 ecDNA 的细胞的动力学。我们还应用这种方法来生成携带含有 Cre 诱导的 Myc 和 Mdm2 的 ecDNA 的小鼠，类似于人类癌症中发生的 ecDNA。我们表明，工程化的 ecDNA 自发积累在来自这些动物的原代细胞中，促进它们的增殖、永生化和转化。最后，我们证明了含有 Mdm2 的 ecDNA 在肝细胞癌的本土小鼠模型中促进肿瘤形成的能力。这些发现为 ecDNA 介导的基因扩增在肿瘤发生中的作用提供了见解。我们预计这种方法对于研究 ecDNA 生物学的进一步未解决的方面以及开发具有特异性局灶性基因扩增的人类癌症的新的临床前免疫活性小鼠模型将很有价值。