Abstract
Stable cell lines that express a gene of specific interest provide an advantage over transient gene expression by reducing variations in transfection efficiency between experiments, sustaining expression for long-term studies, and controlling expression levels in particular if a clonal population is selected. Transient transfection requires introduction of an exogenous gene into host cells via typically harsh chemicals or conditions that permeabilize the cell membrane, which does not normally integrate into the target cell genome. Here, we describe the method of using retroviral transduction to stably express Golgi proteins fused to a promiscuous biotin ligase (TurboID) in HeLa cells, thus creating cell lines that can be leveraged in studies of the proximome/interactome. We also demonstrate a similar protocol for stable expression of a Golgi protein fused to a fluorescent tag via lentiviral transduction. These methods can be further adapted to establish other cell lines with different sub-cellular markers or fusion tags. Viral transduction is a convenient method to create stable cell lines in cell-based studies.