Background

Cell biology studies often require the expression of exogenous or modified proteins (through the use of transgenes) in cells in culture or other model systems, including expression of specific mutants that each selectively perturb a single aspect of that protein’s function. In addition, the study of protein localization often requires expression of the protein of interest fused to a fluorescent protein such as eGFP (Snapp, 2005) or to an enzyme suitable for proximity biotinylation (Gingras et al., 2019). Hence, optimal strategies for expression of transgenes are essential for a wide range of cell biology approaches.

Transient transfection to introduce plasmids encoding these various transgenes into mammalian cells in culture are commonly used for this purpose. However, this strategy can lead to artificial localization or function of the expressed proteins, as the level of expression of the exogenous proteins using this approach often dramatically exceeds that of the endogenous. Furthermore, transient transfection often leads to expression restricted to a minor fraction of cells (5%–20%), which limits the use of this method for cell population–based assays. In contrast, retroviral and lentiviral systems allow for efficient integration of a cassette for expression of a transgene into the genome. When combined with a selection stage, this approach allows for expression of the transgene in a large majority of cells in a population, as well as relatively stable expression levels of the transgene over time. However, the utility of lentiviral or retroviral strategies may be limited by size constraints for the transgene imposed by the packaging process, the time required to generate viral particles, as well as the need for specific biosafety protocols for work with these virus-like particles.

The modification of endogenous genes with CRISPR/Cas9 has also emerged as a compelling strategy to allow expression of specific mutants or fusions of a specific gene from its endogenous promoter, thus avoiding artifacts of protein overexpression (Bukhari and Müller, 2019). While powerful, this strategy also has potential limitations. Generation of loss-of-function mutants of a particular gene using CRISPR/Cas9 involves passaging of these modified cells and may lead to cellular adaptation that may limit the study of the direct effects of the protein being studied. Furthermore, many genes are present in multiple copies in many cultured cell lines, in particular aneuploid cell lines such as those derived from tumors (e.g., the breast cancer cell line MDA-MB-231 used here). Hence, it can be challenging to use CRISPR/Cas9 to modify all copies of a gene within the genome of these cells in culture, limiting the use of this approach for expression of loss-of-function mutants.

The use of transposons for stable integration of transgenes into the genome of cultured cells is an effective strategy that parallels the use of viral transduction strategies, without the need for packaging of transgene material into viral particles. The Sleeping Beauty transposon method enables stable integration of a transgene found within a vector containing inverted terminal repeats (ITRs) when co-transfected into cells with a vector encoding a transposase enzyme. This approach can be used in combination with selection to achieve integration of the transgene into a large majority of cells in a population (Kowarz et al., 2015).

While the Sleeping Beauty system has been used extensively, a re-engineered transposase enzyme with enhanced activity—SB100X (Mátés et al., 2009)—is ideally suited for this approach, and achieves highly efficient and random integration into TA dinucleotide sites within the target cell genome (30,000 such sites in the human genome) (Kowarz et al., 2015). Kowarz & col. developed a suite of plasmids for use of the Sleeping Beauty transposon system that includes plasmids to allow either inducible (pSBtet) or constitutive (e.g., pSBbi) expression and a range of fluorescent protein and antibiotic selection markers and demonstrated the use of these strategies for stable gene expression in HeLa and HEK293T cells (Kowarz et al., 2015). The pSBtet series of plasmids developed by Kowarz & col. allows doxycycline-inducible expression of a transgene alongside constitutive expression of one of several selection markers (puromycin, hygromycin, neomycin, or blasticidin) and a fluorescent protein (BFP, eGFP, or RFP). We focus here on the use of pSBtet-BP (constitutive expression of BFP and puromycin selection, Figure 1A). Notably, the selection marker, fluorescent protein, and reverse tetracycline transactivator (rtTA) in the pSBtet plasmids are expressed as a self-cleaving polyprotein (Figure 1A).

Figure 1. pSBtet-BP plasmid and cloning strategy. (A) Map of the pSBtet-BP plasmid as obtained from Addgene, plasmid number 60496 and developed by Kowarz & col. (Kowarz et al., 2015). Shown are the Tetracycline responsive element (TRE) promoter, the open reading frame encoding luciferase (which is replaced by the transgene of interest during the cloning stage, Procedure A), as well as the expression of a polyprotein from the constitutive synthetic RPBSA promoter; this polyprotein undergoes cleavage to generate blue fluorescent protein (BFP), reverse tetracycline transactivator (rtTA), and puromycin resistance protein. Also shown is the expression of ampicillin resistance for bacterial selection. Also shown (dashed line boxes) are the regions relevant to subcloning of the transgene, as described in Procedure A. (B) Shown is the cloning strategy for excision of the luciferase-encoding sequence and subcloning of the transgene of interest using NcoI and ClaI sites within the pSBtet-BP plasmid. NcoI and ClaI cut sites are shown in red text; the Kozak sequence is shown in yellow and the open reading frame (ORF) of the luciferase is underlined.

We recently used the Sleeping Beauty transposon strategy developed by Kowarz & col. (Kowarz et al., 2015) in ARPE-19, MDA-MB-231, and SUM149-PT cells for generation of stable cells that allow doxycycline-inducible expression of protein fusions or mutants (Cabral-Dias et al., 2022; Rahmani et al., 2023; Sugiyama et al., 2023) or short hairpin RNA (shRNA) (Lo et al., 2023). Here, we first describe subcloning of a transgene of interest into the pSBtet plasmid (Procedure A). We also note that the use of these Sleeping Beauty plasmids requires an initial transfection and selection process that is unique to some cell lines. We thus report the protocol optimized for the generation of stable cells in ARPE-19 and MDA-MB-231 (Procedure B). Finally, the use of doxycycline for expression of a transgene by the Sleeping Beauty strategy can allow for control of expression to approximate the endogenous level of expression of the protein. This is achieved through titration of doxycycline, allowing determination of an optimal range of doxycycline for each stable cell line generated. This approach allows expression of the transgene at near-endogenous levels, either alone or in combination with siRNA gene silencing of the endogenous protein for knockdown-rescue approaches (Cabral-Dias et al., 2022; Rahmani et al., 2023; Sugiyama et al., 2023). Thus, we also describe the use of doxycycline to induce expression of the protein of interest, either alone to determine optimal doxycycline condition for expression of the transgene (Procedure C), or in combination with siRNA gene silencing of the endogenous protein for knockdown-rescue approaches (Procedure D).