Stop-Codon–Mediated Polycistronic Translation Enables Multi-ORF Expression in Mammalian Neurons In Vivo

Abstract Multigene expression in mammalian systems remains constrained by the presumed monocistronic nature of translation and by reliance on bulky regulatory elements such as IRESs or self-cleaving peptides. Here we show that canonical stop codons alone are sufficient to drive robust polycistronic protein expression in mammals. We demonstrate efficient bi- and tricistronic expression of full-length, functional open reading frames from a single mRNA, without any auxiliary translational elements, linkers, or sequence modifications. This strategy operates across diverse protein classes and supports downstream translation over intercistronic distances exceeding 2 kb. Crucially, we achieve tricistronic expression in mammalian neurons in vivo following adeno-associated virus delivery to the rat brain. To our knowledge, this is the first demonstration of in vivo expression of three independent protein-coding ORFs from a single mammalian transcript using only canonical stop codons. Leveraging this capability, we introduce Genetically Encoded Functional Rainbow Indicators (GEFRIs), compact tricistronic reporters enabling simultaneous structural labeling and functional calcium imaging from a single viral vector. Together, this work establishes stop-codon–mediated polycistronism as a practical, scarless, and uniquely compact platform for coordinated multi-protein expression in mammalian cells and in vivo .