Background

The introduction of plasmid DNA into mammalian cells has been an essential method to support cellular and molecular biology investigations. Typically, the introduction of plasmid DNA can be mediated by a few approaches, including electroporation, chemical transformation, polyfection using polymer-based transfection reagents, microinjection, and viral transduction. These methods vary in efficiency depending on cell type and scale. Another factor to consider is whether the researcher wants transient over-expression of a gene of interest or generation of stably transfected cell lines. The generation of a stable cell line is typically more time-consuming. Still, it can be advantageous if cells are refractory to lipofection or if the same cell lines will be used for several experiments. In our lab, we use the Sleeping Beauty transposon system to help facilitate the integration of DNA into the genome to support the generation of stable cell lines [2–4]. This transposon system has been widely used as a genetic engineering tool for the last decade. Indeed, we have used this system to generate RAW264.7 cells expressing the reverse tetracycline repressor (rTetR) for controllable induction of our protein of interest, TagBFP-INPP4B-CaaX [5]. As these cells are generally challenging to transfect transiently, this approach allows for the rapid creation of cell lines.

The protocol described here is an adaptation of Kowarz et al. [1]. Briefly, this system utilizes a plasmid (SB100x) encoding the Sleeping Beauty transposase and a second plasmid containing an integration cassette with a gene of interest, a selectable marker, flanked by inverted terminal repeats. Kowarz et al. [1] generated a variety of plasmids, available from Addgene, to allow researchers to create cells with constitutive or doxycycline-inducible genes, an array of drug-resistant markers (e.g., puromycin or hygromycin) and the option also to deliver the reverse tetracycline repressor.

Phagocytosis is an actin-driven process used when cells ingest particles greater than 0.5 mm, such as pathogens and apoptotic bodies [6–8]. Phagocytosis entails the extension of actin-rich pseudopods to surround the target and pull the particle into the cell, forming a nascent phagosome [9]. The extensive actin remodeling, polymerization, bundling, and depolymerization must be exquisitely controlled and temporally coordinated. Major regulators in this process are the metabolism and interconversion of phosphoinositides lipids [10]. For instance, the generation of phosphatidylinositol (3,4)-bisphosphate (PtdIns3,4P₂) at the engagement site is crucial for extending pseudopods to envelop prey [5]. Mammalian cells contain at least two pathways and multiple enzymes capable of synthesizing PtdIns3,4P₂; thus, the knockdown of individual enzymes or specific inhibitors often generates unsatisfying results [11]. Instead, we used a synthetic plasmid-borne enzyme inositol polyphosphate 4-phosphatase type IIB (INPP4B) produced as a chimera with TagBFP for visualization, as well as a C-terminal CaaX motif for prenylation and membrane targeting [12]. This chimeric protein catalyzes the dephosphorylation of the D-4 position of PtdIns3,4P₂ in the plasma membrane regardless of the biosynthetic route [12].

We generate doxycycline-inducible RAW264.7 cells expressing BFP-INPP4B-CaaX and a second cell line expressing the catalytically inactive BFP-INPP4B^C842A-CaaX that serves as a control. Following overnight treatment with doxycycline to induce the INPP4B-containing constructs, we conduct phagocytosis assays to determine the role of PtdIns3,4P₂ [5]. Typically, our phagocytosis assays use IgG-opsonized sheep red blood cells or IgG-coated polystyrene beads as prey to assess Fcg receptor-mediated phagocytosis [13].

Many approaches are available to assess phagocytosis; most rely on fluorescent labels on the prey to quantify internalization and phagosome maturation. Flow cytometry analysis determines the overall ingestion of particles by measuring total fluorescence intensity/cell as a parameter for particle uptake [14]. Other methods use pH-sensitive fluorescent particles that become brighter in acidic conditions, such as in the phagolysosome, that measure particle uptake and phagosome maturation [15]. However, both these approaches are limited in defining particle engagement vs. complete engulfment. Instead, we prefer a high-resolution confocal microscope and an antibody staining approach to distinguish bound vs. fully internalized particles (Figure 1).

Figure 1. INPP4B-CaaX inhibits the internalization of IgG opsonized particles. A. Representation of the assay. All sheep red blood cells (SRBCs) are labeled with the AF488-conjugated antibody, whereas only extracellular SRBCs are stained with the AF647 antibody at the end of the incubation period. B. Representative image. Fluorescence markers are pseudocolored: AF488 (green), AF647 (magenta), and BFP (cyan). Scale bar = 5 μm. C. A double y-axis graph depicting the results of panel B. The phagocytic index represents the number of beads internalized by the cells over a fixed period. Phagocytic efficiency represents the percentage of engaged particles that are internalized.