Generating actb-MBS cell line stably expressing NLS-MCP-HaloTag and Dendra2-RPB1

We used a previously engineered mouse embryonic fibroblasts (MEF) cell line in which a cassette containing 24× MS2 binding sites (MBS) is knocked in the 3´ untranslated region (UTR) of the endogenous β-actin gene (Actb-MBS cells) (Lionnet et al., 2011a). In order to generate stable expression of the MS2 capsid protein (MCP) fused to a HaloTag moiety, we incubated the Actb-MBS cells with lentiviral particles generated using the pHAGE-Ubc-NLS-MCP-HaloTag vector. Five days after infection, we stained the cells with the Janelia Fluor 549 (JF549) HaloTag ligand (Grimm et al., 2015) by incubating them 15 min at 37°C in growth medium (DMEM, 10% FBS) supplemented with 100 nM fluorescent ligand, and then washed the unbound ligand (15 min in fresh growth medium at 37°C, followed by 2 washes in fresh growth medium). Immediately after staining, JF549-positive cells were sorted using flow cytometry.

In order to generate stable expression of the Dendra2-RPB1Amr construct in our cell line, Actb-MBS cells stably expressing NLS-MCP-HaloTag were transfected with the piggyback vector (PB53x EF1-Dendra2-RPB1Amr) along with a plasmid expressing the super Piggybac Transposase using an Amaxa nucleofector (Lonza, Basel, Switzerland). Cells expressing Dendra2-RPB1-Amr were then selected with alpha-Amanitin (2 ug/mL, Sigma-Aldrich, St. Louis, MO) for 2 weeks, starting 2 days post-transfection (Cisse et al., 2013). After the 2-week drug-selection period, cells were stained with the Janelia Fluor 646 (JF₆₄₆) ligand (Grimm et al., 2015), using the same protocol as JF₅₄₉ (see above). Double positive cells (green fluorescence from Dendra2; red fluorescence from JF₆₄₆) were sorted using flow cytometry. Cells were derived in-house from primary MEFs. Following immortalization and integration of fluorescent labels, cell identity was regularly controlled by visualizing the fluorescence in the Dendra2 and MCP-HaltoTaq channels (assaying correct nuclear localization for both proteins, photo-conversion upon 405 excitation for Dendra2, and presence of hundreds of mRNA particles in the MCP-HaloTaq channel). The cell lines undergo regular mycoplasma contamination testing by the Janelia Cell Culture Facility and also at the Massachusetts Institute of Technology.