Immortalization and transformation of primary cells mediated by engineered ecDNAs.

Focal gene amplifications are among the most common cancer-associated mutations, but their evolution and contribution to tumorigenesis have proven challenging to recapitulate in primary cells and model organisms. Here we describe a general approach to engineer large (>1 Mbp) focal amplifications mediated by extrachromosomal circular DNAs (ecDNAs, also known as "double minutes") in a spatiotemporally controlled manner in cancer cell lines and in primary cells derived from genetically engineered mice. With this strategy, ecDNA formation can be coupled with expression of fluorescent reporters or other selectable markers to enable the identification and tracking of ecDNA-containing cells. We demonstrate the feasibility of this approach by engineering MDM2-containing ecDNAs in near-diploid human cells, showing that GFP expression can be used to track ecDNA dynamics under physiological conditions or in the presence of specific selective pressures. We also apply this approach to generate mice harboring inducible Myc - and Mdm2 -containing ecDNAs analogous to those spontaneously occurring in human cancers. We show that the engineered ecDNAs rapidly accumulate in primary cells derived from these animals, promoting proliferation, immortalization, and transformation.

Surgical Methods in Postmetamorphic Xenopus laevis: Optic Nerve Crush Injury Model.

Many human optic neuropathies lead to crippling conditions resulting in partial or complete loss of vision. While the retina is made up of several different cell types, retinal ganglion cells (RGCs) are the only cell type connecting the eye to the brain. Optic nerve crush injuries, wherein RGC axons are damaged without severing the optic nerve sheath, can serve as a model for traumatic optical neuropathies as well as some progressive neuropathies such as glaucoma. In this chapter, we describe two different surgical methods for establishing an optic nerve crush (ONC) injury in the postmetamorphic frog, Xenopus laevis. Why use the frog as an animal model? Mammals lose the ability to regenerate damaged CNS neurons, but amphibians and fish retain the ability to regenerate new RGC bodies and regrow RGC axons following an injury. In addition to presenting two different surgical ONC injury methods, we highlight their advantages and disadvantages and discuss the distinctive characteristics of Xenopus laevis as an animal model for studying CNS regeneration.