Hygromycin B is an antibiotic used by scientists in bio-research to select cells modified by genetic engineering. It inhibits the growth of a wide range of prokaryotic and eukaryotic cells by interfering with protein synthesis. Hygromycin B allows the selection and maintenance of cells expressing the hygromycin-resistance gene.
THE RESEARCH STUDY
The hygromycin phosphotransferase gene was fused in-frame with the herpes simplex virus type 1 thymidine kinase gene. The resulting fusion gene (termed HyTK) confers hygromycin B resistance for dominant positive selection and ganciclovir sensitivity for negative selection and provides a means by which these selectable phenotypes may be expressed and regulated as a single genetic entity.
A variety of genes confer positive selectable phenotypes on mammalian cells (4, 8, 17, 20, 28, 29). Such genes allow enrichment of cells that have retained introduced genes in a stable form, thereby facilitating gene transfer studies. Generally, dominant selectable genes, such as the hygromycin phosphotransferase gene (20), are more versatile. Unlike recessive selectable genes, such as the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene (29), the use of which is limited to mutant cells deficient in the selectable function, dominant selectable genes may be used in wild-type cells.
Several genes confer both negative and positive selectable phenotypes (2, 5, 22, 23). For example, HSV-1 TK efficiently converts ganciclovir (GCV) into a cytotoxic nucleotide analog, but GCV is a poor substrate for mammalian TK (22). Thus, GCV may be used to select against wild-type cells expressing the HSV-1 TK gene (22). Although the HSV-1 TK gene may be used for positive selection, this is effectively limited to mutant cells lacking TK activity (29). Use of the hypoxanthine phosphoribosyltransferase (HPRT) gene (8) or the adenine phosphoribosyltransferase (APRT) gene (28) for either positive or negative selection is similarly limited to HPRT- or APRT- cells, respectively (5, 23). Thus, we sought to create a new gene that could be used for positive and negative selection in wild-type cells. This was accomplished by fusing the hph gene in-frame with the HSV-1 TK gene. The hygromycin phosphotransferase and HSV-1 TK genes were first placed under the regulatory control of the human cytomegalovirus (HCMV) IE94 promoter in tgCMV/hygro and tgCMV/TK, respectively (Fig. 1A). The hph-TK gene fusion was constructed by inserting the 1,644-bp SpeI-ScaI fragment from tgCMV/hygro between the SpeI and MluI sites of tgCMV/TK to create tgCMV/HyTK (Fig. 1A). Before ligation, the MluI site in the HSV-1 TK gene was treated with T4 DNA polymerase to allow blunt-end ligation with the Scal site, thus preserving the open reading frame.
Translation of this fused gene (designated the HyTK selectable fusion gene) would be expected to generate a bifunctional fusion protein. To demonstrate that the HyTK selectable fusion gene encodes both hygromycin phosphotransferase and HSV-1 TK enzymatic activities, tgCMV/HyTK was transfected into NIH 3T3 and Rat-2 cells (25), and the transfected cells were selected in hygromycin B or hypoxanthine-aminopterin-thymidine (HAT). Expo-nentially dividing cells were harvested by trypsinization, washed free of serum, and resuspended in serum-free medium at a concentration of 107 cells per ml. Supercoiled plasmid DNA (5 t.g) was added to 800 ,ul of cell suspension (8 x 106 cells), and the mixture was electroporated by using the Bio-Rad Gene Pulser (200 to 300 V, 960 ,uF, 0.4-cm electrode gap, at ambient temperature). The cells were then returned to 9-cm dishes and grown in nonselective medium. After 24 h, the transfected cells were trypsinized, seeded at a density of 6 x 105 cells per 9-cm dish, and allowed to attach overnight. The medium was supplemented with hygromycin b (500 p.g/ml for NIH 3T3 cells and 300 ,ug/ml for Rat-2 cells) or HAT, and selection was continued for 10 to 12 days. The drug-resistant colonies were then fixed in situ with 100% methanol, stained with methylene blue, and enumerated. The results shown in Table 1 demonstrate that when transfected into NIH 3T3 and Rat-2 cells, the HyTK selectable fusion gene conferred both resistance to hygromycin B (Hmr and the ability to grow in HAT medium (HATr)).
Sources: STEPHEN D. LUPTON, LAURIE L. BRUNTON, VICTORIA A. KALBERG, AND ROBERT W. OVERELL