Dominant positive and negative selection using luciferase, green fluorescent protein and beta-galactosidase reporter gene fusions.

The introduction of exogenous DNA sequences into cultured cells is a cornerstone in the study of mammalian gene expression. For most cell types, the efficiency of stable gene transfer is usually quite low, which mandates the use of selectable markers to isolate a population of transfected cells with the desired phenotype. In simple gene expression experiments, only one type of selectable marker is required. Positive selectable markers such as the bacterial neo and hph genes facilitate the direct selection for cells that are resistant to the cytocidal effects of the antibiotics, G418 and hygromycin B, respectively (1,12). Conversely, negative selectable markers facilitate the elimination of the cells in which they are expressed. The most popular negative selectable marker is the herpes simplex virus thymidine kinase gene, tk, which sensitizes cells to the toxic effects of the nucleoside analogue gancyclovir (11,15). In more complex gene transfer experiments such as in the use of gene, promoter or enhancer traps, it becomes necessary to include multiple marker genes. For example, fusions between the bacterial lacZ reporter gene and the selectable neo marker (2), the hph and tk selectable markers (7) or the neo and tk selectable markers have been developed for use in enhancer or gene trapping experiments. Here, we describe the further development of gene fusions between the tk and neo selectable markers and the bacterial lacZ, jellyfish gfp and firefly luc reporter genes. The gene fusions were constructed using cDNAs derived from the previously described pPNT (14), pβgeo (2), pGL3-Control (Promega, Madison, WI, USA), and pEGFP-C1 (Clontech Laboratories, Palo Alto, CA, USA) vector constructs, which encode the HSV tk and bacterial neo genes, a lacZ-neo fusion gene, the enhanced luc and gfp reporter genes, respectively. A tkneo gene fusion was generated using PCR amplification in conjunction with vector-specific primers to eliminate the endogenous stop codon in the tk gene of pPNT through the incorporation of a BamHI site. The amplified product was cloned as an EcoRI-BamHI fragment into the corresponding EcoRI-BamHI sites of pβgeo, which effectively replaces the lacZ-portion of the lacZ-neo gene fusion. The tkneo fusion gene was then subcloned as a HindIII-NotI fragment into the pEFG3 expression vector, and this construct was designated pEF-tkneo (Figure 1). We incorporated the gfp cDNA by subcloning the corresponding reading frame as a blunt-ended NheIBglII fragment from pEGFP-C1 into the blunt-ended BamHI site to generate pEF-tkgfneo. The tklucneo gene fusion was generated using PCR amplification in conjunction with vector-specific primers to introduce PacI restriction endonuclease sites into the 5′-UTR and to eliminate the endogenous stop codon in the luc gene of pGL3-Control, which was then subcloned into the BamHI site of pEFtkneo to generate pEF-tkβgeo (Figure 1). The lacZ gene was incorporated into the tkneo gene fusion by subcloning the corresponding reading frame as a BamHI fragment from pβgeo into the BamHI site of pEF-tkneo to generate pEF-tkβgeo (Figure 1). The gfp cDNA was incorporated into the BamHI site of this construct to generate pEFTβGN (Figure 1). As positive control plasmids for our experiments, we subcloned the individual luc, gfp and lacZ genes into the pEF1 vector (Invitrogen, Carlsbad, CA, USA) to generate pEFLuc, pEF-GFP and pEF-βGal, respectively (Figure 1). The activity of the reporter gene components in each of the five fusion genes was assayed following transient transfection into HEK293 (human embryonic kidney) cells. Cells were grown in DMEM supplemented with Benchmarks