Proc. Natl. Acad. Sci. USA Vol. 88, pp. 10416-10420, December 1991 Genetics A dominant positive and negative selectable gene for use in mammalian cells (gene fusion/herpes simplex virus thymidine kinase/bacterial neomycin phosphotransferase) FAINA SCHWARTZ*t, NOBUYO MAEDAt, OLIVER SMITHIESt, ROBERT HICKEY§¶, WINFRIED EDELMANNII ARTHUR SKOULTCHI§, AND RAJU KUCHERLAPATIII *Department of Genetics, University of Illinois College of Medicine, Chicago, IL 60612; tDepartment of Pathology, University of North Carolina, Chapel Hill, NC 27599; and Departments of §Cell Biology and I"Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461 Contributed by Oliver Smithies, August 23, 1991 ABSTRACT We have constructed three different fusion selected in a negative manner. Unfortunately, no single gene genes containing the herpes simplex virus thymidine kinase is available that can be selected for and against in wild-type (HSV tk) and the bacterial neomycin phosphotransferase (neo) cells. The availability of such genes would be valuable. genes. All three fusion genes utilize the HSV tk promoter but To achieve this goal, we have constructed and tested fusion differ at thejunction oftheir components. We have determined genes between the bacterial neo and HSV tk genes. The neo if the fusion genes are bifunctional by introducing them into serves as a positive selectable marker and the tk serves as a mammalian cells and testing for function of the individual negative selectable marker. We here report experiments components. One of the fusion genes, TNFUS 69, produced a showing that this type of bifunctional gene can be used as a bicistronic message and a fusion protein that has TK and NEO dominant positive and negative selectable marker in mam- protein functions. This and other fusion genes of a similar malian cells. nature could serve as dominant positive and negative selectable markers in mammalian cells. MATERIALS AND METHODS Gene transfer into somatic mammalian cells as well as into Cells. Mouse LMTK- cells (6) were maintained in Dulbec- fertilized mouse embryos has become an extremely valuable co's modified Eagle's medium (DMEM, GIBCO) supple- tool for study of gene structure-function relationships. Gene mented with 10% fetal calf serum (FCS). Mouse embryonic transfer can be accomplished by several different methods stem (ES) cells were maintained on irradiated feeder layers of that include microinjection, calcium phosphate coprecipita- STO cells in DMEM/10% FCS and 2% 2-mercaptoethanol. tion, and electroporation. With the exception of direct mi- Transfections. LMTK- cells were transfected by the cal- croinjection of DNA into the nuclei of recipient cells, these cium phosphate coprecipitation method (7). For each trans- methods require the presence ofa selectable gene in the input fection, 5-7 x 105 cells were plated in 60-mm culture dishes DNA to permit isolation ofcells that have stably incorporated and incubated overnight. A calcium phosphate precipitate the exogenously introduced DNA. Early gene transfer ex- with the appropriate DNA was added to each plate. No periments utilized cells mutant at the thymidine kinase (tk) carrier DNA was used. After 4 hr, the cells were treated for locus, and a cloned herpes simplex virus (HSV) tk gene was 2 min with 10% dimethyl sulfoxide in 138 mM NaCl/5 mM used as a directly selectable marker in transfecting these cells KCl/0.7 mM Na2HPO4/6 mM dextrose/20 mM Hepes, pH (1). Subsequent experiments allowed introduction of nonse- 6.92. Mouse ES cells were transfected by electroporation lectable DNA sequences by ligating them to the selectable tk using a Bio-Rad gene pulser at 500 kF and 250 V. The or by cotransfection (2). Although other selectable genes, transfected cells were incubated overnight in DMEM con- such as the hypoxanthine phosphoribosyltransferase (hprt), taining 10% FCS, after which they were transferred to have been introduced into appropriate mutant mammalian 100-mm culture dishes and subjected to selection with G418 cells, the need for the target cell to be mutant has limited the at 200 jig/ml (Geneticin, GIBCO) or hypoxanthine (15 jkg/ types of cells that could be used as recipients. The develop- ml), aminopterin (0.2 mg/ml), and thymidine (5 tug/ml) (HAT ment of dominant selectable systems has removed this lim- medium; ref. 8). Resistant colonies were counted after 14 itation. The first such system used was the xanthine guano- days of selection. Individual colonies were isolated and sine phosphoribosyltransferase gene (gpt) from Escherichia expanded for further analysis. coli (Eco-gpt; ref. 3). The gpt expression could be selected for Preparation of Cell Extracts. Cell monolayers were washed in hprt- or in wild-type cells. Other dominant selectable with phosphate-buffered saline, harvested, and centrifuged at genes have been developed, among which the neomycin 2000 x g. The cell pellet was suspended in cell lysis buffer phosphotransferase (nptII or neo) is perhaps the most widely (0.1 M Tris, pH 6.8/0.5% Triton X-100/1 mM phenylmeth- used (4). ylsulfonyl fluoride) at 108 cells per ml. Cells were sonicated The ability to apply positive and negative selection systems three times, 10 sec each time, and cell lysis was monitored by during the introduction ofDNA into cells has proven to be an phase-contrast microscopy. Lysed cells were centrifuged for extremely valuable genetic tool (5). The most widely used 2 min to sediment cell membranes, nuclei, and cell debris, genes that allow positive and negative selection schemes are and the supernatant was removed for protein analysis. Pro- hprt, tk, and adenine phosphoribosyltransferase (aprt). By using appropriate mutants as recipients, transformed cells Abbreviations: HSV, herpes simplex virus; neo, neomycin phos- that have acquired the gene can be selected positively. Cells photransferase; tk, thymidine kinase; SV40, simian virus 40; R. that have expression of these genes can subsequently be resistant; ES, embryonic stem; HAT, hypoxanthine/aminopterin/ thymidine; nt, nucleotide(s). tPresent address: Genetics Division, Children's Hospital, Boston, The publication costs of this article were defrayed in part by page charge MA 02115. payment. This article must therefore be hereby marked "advertisement" Present address: Department of Pharmacology, University of Mary- in accordance with 18 U.S.C. §1734 solely to indicate this fact. land School of Medicine, Baltimore, MD 21201. 10416 Genetics: Schwartz et al. Proc. Natl. Acad. Sci. USA 88 (1991) 10417 tein concentration was estimated by the Bio-Rad protein Denhardt's = 5 g of Ficoll, 5 g of polyvinylpyrrolidone/5 g of assay. bovine serum albumin and H20 to 500 ml)/0.1% sodium Gel Electrophoresis and Western Blot Analysis. Duplicates dodecyl sulfate (SDS)/60 Ag of denatured salmon sperm of each sample were analyzed by SDS/polyacrylamide gel DNA per ml. The filters were washed at 550C with 0.1 x electrophoresis, using 80-100 pug of total protein per lane SSC/0.1% SDS. The filters were exposed to Kodak XAR film according to the procedure of Laemmli (9). Electrophoretic to generate autoradiographs. transfer ofproteins and antibody binding were carried out by a modification of the protocol of Towbin et al. (10). Proteins were transferred to nitrocellulose filter paper overnight at 200 RESULTS mA in 25 mM Tris, pH 8.1/192 mM glycine made in 20% Nature of the Test Plasmids. The HSV tk gene was derived (vol/vol) methanol. After transfer, the nitrocellulose filter from pHSV106 (13) and the neo gene was derived from was cut in half and soaked in a solution of 3% bovine serum pSV2neo (4). Using plasmid pSVO10 (4) as a vector, an initial albumin (fraction V, Sigma) in saline (0.9% NaCl/10 mM plasmid was made that contained the tk gene ofthe HSV type Tris-HCl, pH 7.4) for 1 hr at room temperature. One half of I (HSV I tk) together with its own promoter and all of the the nitrocellulose filter was incubated with goat antiserum necessary 5' regulatory sequences (14); a promoterless copy prepared against the TK protein ofthe HSV type I (a gift from ofthe bacterial aminoglycoside phosphotransferase gene neo T. Silhavy, Princeton University, Princeton, NJ). The sec- (15) was inserted downstream of the tk gene. This initial ondary antibody was rabbit anti-goat IgG (a gift from W. plasmid also contained the simian virus 40 (SV40) replication Carey Hanly, University of Illinois, Chicago) diluted 1:1000 origin and the SV40 early promoter regions in reverse ori- in the above buffer. The second halfofthe nitrocellulose filter entation with respect to the tk gene; its structure is illustrated was incubated with primary rabbit antiserum prepared in the top line of Fig. 1. Several modifications of the initial against bacterial neo (anti-NEO antibody; a gift from L. plasmid were made in the tk and neo genes resulting in three Shapiro, Stanford University, Palo Alto, CA). After each different constructs designated TNT, TNSSS 31, and TN- incubation, filters were extensively washed in a solution of FUS 69. The regions corresponding to the end ofthe tk coding 0.05% Tween 20 prepared in Tris-buffered saline. Immuno- sequences and beginning of the neo in the initial plasmid and reaction was visualized using goat anti-rabbit immunoglob- in each of these three constructs are also shown in Fig. 1. In ulins coupled to horseradish peroxidase (Bio-Rad) and stain- the TNT (tk-neo terminator) plasmid, the region correspond- ing with horseradish peroxidase color development reagent ing to last 7 amino acids of the TK protein was replaced by according to manufacturer's specifications. The developed a region coding for 6 new amino acids. The end of the TK membrane shows purple bands against a white background. protein and the initiation codon for the NEO protein are The sensitivity of detection by this procedure is 100 pg for a separated by 12 nucleotides (nt).