Proc. Natl. Acad. Sci. USA Vol. 84, pp. 8463-8467, December 1987 Cell Biology Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading (gene expression) MARCUS FECHHEIMER*, JOHN F. BOYLANt, SANDRA PARKER*, JESSE E. SISKENt, GORDHAN L. PATEL*, AND STEPHEN G. ZIMMERt *Department of Zoology, University of Georgia, Athens, GA 30602; and tDepartment of Medical Microbiology and Immunology, University of Kentucky Medical Center, Lexington, KY 40536 Communicated by J. Herbert Taylor, August 6, 1987 (receivedfor review March 19, 1987) ABSTRACT Scrape loading and sonication loading are living cells, and we discuss the potential utility of these two recently described methods ofintroducing macromolecules methods as supplements to the existing arsenal of methods into living cells. We have tested the efficacy of these methods for obtaining cellular expression of sequence information for transfection ofmammalian cells with exogenous DNA, using present on molecules of exogenous DNA. selection systems based either on resistance to the drug G418 (Geneticin) or on acquisition of the ability to utilize the salvage pathway of pyrimidine biosynthesis. These loading methods MATERIALS AND METHODS can be employed to generate cell lines that express the gene product of the transfected DNA molecules both transiently and Materials. Dulbecco's modified Eagle's medium, fetal stably. Optimal transfection is observed when the DNA is bovine serum, trypsin, penicillin, streptomycin, Geneticin added to cells in physiological saline lacking divalent cations (G418 sulfate), and Fungizone were supplied by GIBCO. and containing K+ in place of Na'. DNA molecules 7.1 to 30 Hypoxanthine, aminopterin, thymidine, and fluorescein- kilobases long have been introduced by the scrape loading labeled dextran (FD-40) were obtained from Sigma. Murine procedure. In addition, the scrape loading procedure has been fibroblasts deficient in the enzyme thymidine kinase (LTK-) employed for cotransfection and subsequent expression of and the pPVTK4 plasmid, which contains the thymidine nonselectable genes encoded on DNA molecules added in a kinase gene in pBR328, were kindly supplied by Peter mixture with DNA molecules whose expression is selected. Cell Stambrook (University of Cincinnati). Plasmids were grown lines expressing oncogenes or proteins that are important for in Escherichia coli and the DNA was purified by cesium regulation of cell growth and division have been obtained by chloride gradient centrifugation in the presence of ethidium this procedure. The scrape loading procedure is also useful for bromide as described (25). Adenovirus 2 DNA and pSV2neo studies of the cellular changes that occur upon expression of an (26) were purchased from Bethesda Research Laboratories. exogenous gene. As many as 80% ofcells scrape loaded with the HeLa cells, monoclonal antibody (PAB101) reactive with the plasmid pC6, which encodes the simian virus 40 large tumor simian virus 40 (SV40) large tumor (T) antigen (TIB117), and antigen, contained this protein in the nucleus between 1 and 5 c-Ha-ras DNA were purchased from the American Type days after transfection. Thus, scrape loading and sonication Culture Collection. A 6.6-kilobase (kb) BamHI fragment loading are simple, economical, and reproducible methods for containing the ras oncogene was inserted into the BamHI site introduction of DNA molecules into adherent and nonadherent of pBR322. Plasmid pC6, which encodes the SV40 large T cells, and these methods may be useful in the future for antigen, was obtained from Yakov Gluzman (Cold Spring experimentation at both fundamental and applied levels. Harbor Laboratories). The insert was at the EcoRI site of pMKI6 #6. Rat embryo fibroblasts (REF) were produced as Methods for introduction of foreign DNA into living cells in described (27) and WT/O-15.5 D2 (abbreviated D2) cells a form allowing its expression in the recipient cell have been were obtained from Paul Fisher (Columbia University). The widely used in development of human gene therapy technol- murine myeloma cell line P3-X63-Ag8.653 (28) was provided ogies (1), in strategies for plant genetic engineering (2), in by Dan Murfin (University of Georgia). improvement of domestic livestock (3, 4), and in basic Cell Growth. HeLa cells, hepatic tissue culture cells, and research. A number of methods for introduction ofDNA LTK- fibroblasts were grown in Dulbecco's modified eagle's into medium supplemented with 10% fetal bovine serum, penicil- living cells are currently in use (5). These techniques include lin at 50 units/ml, streptomycin at 50 A.g/ml, and Fungizone direct microinjection (6, 7), precipitation with calcium phos- at 2.5 jug/ml (complete medium). The murine myeloma phate (8, 9), membrane breakdown induced by transient P3-X63-Ag8.653 was grown in the same medium containing electric fields (10-12), fusion of erythrocytes or liposomes 50 ,uM 2-mercaptoethanol. REF and D2 cells were grown in (13-16), osmotic lysis of pinosomes (17), centrifugation plastic tissue culture flasks (Corning), using Dulbecco's loading (18), delivery of high-velocity microprojectiles (19), modified Eagle's minimal essential medium supplemented retroviral vectors (20, 21), and the natural plant vector with 10o fetal bovine serum, penicillin at 50 units/ml, Agrobacterium tumefaciens (22). streptomycin at 10 pmg/ml, 0.3% sodium bicarbonate, 1 mM Two additional methods for introduction of macromol- glutamine, and 2.5 mM Hepes. ecules into living cells, scrape loading (23) and sonication Introduction of Thymidine Kinase Gene into LTK- Fibro- loading (24), have recently been described. In this report, we blasts by Scrape Loading and Sonication Loading. In the describe the application of scrape loading and sonication scrape loading procedure, culture medium was removed from loading to the introduction offunctional genetic material into a 10-cm Petri dish with a nearly confluent monolayer of LTK- cells. The cell monolayer was then washed once with The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: SV4Q, simian virus 40; T antigen, tumor antigen; in accordance with 18 U.S.C. §1734 solely to indicate this fact. REF, rat embryo fibroblasts. 8463 Downloaded by guest on September 24, 2021 8464 Cell Biology: Fechheimer et al. Proc. Natl. Acad. Sci. USA 84 (1987) 5 ml of 10 mM KH2PO4/1.0 mg of glucose per ml/0.1 mM dextran by using either the scraping or the sonication proto- EDTA/140 mM NaCl, pH 7.2 (loading solution). pPVTK4 col. Fluorescein-labeled dextran was added to cells at a final DNA (0-50 Ag/ml) in 1 ml of the loading solution was added concentration of 10 mg/ml, and cells were then treated as to the cells. The cells were then loaded with DNA by scraping described above. Cells exhibiting cytoplasmic fluorescence them from the dish by using a rubber policeman as previously were enumerated by examination of the living cells by described (23). Cells were counted by using a hemocytome- fluorescence microscopy both 6 and 24 hr after loading. ter, and viable cells were identified by exclusion of the dye Control experiments included removal of cells from the trypan blue. In some experiments, variations in the loading substrate by trypsin digestion in the presence of fluorescein- solution were employed in which KCI was used in place of labeled dextran and addition of the fluorescein-labeled dex- NaCl and in which the solutions were supplemented with 2 tran to the cells after scraping or sonication treatments had mM MgC12. been performed. Little or no loading was observed in these In the sonication loading procedure, after removal of control experiments. medium from a nearly confluent monolayer of LTK- fibro- Quantification of Transient Expression by Immunofluores- blasts in a 10-cm dish, the cells were washed with 5 ml of cence Microscopy. REF cells were loaded by scraping with 10 Dulbecco's phosphate-buffered saline without Ca2+ and gg of plasmid pC6 DNA (SV40 large T antigen) in 0.2 ml of Mg2+ (PBS) and removed from the substrate by trypsin PBS. Scraped cells were plated in four-well chamber slides, digestion. The cells were diluted into 2 ml of complete fixed 0, 1, 2, 3, 4, or 5 days later, and stained with antibody medium, sedimented, resuspended in 10 ml of loading solu- reactive with the SV40 large T antigen to detect expression tion, and resedimented. The cells were then suspended in 1 of the transformed gene product as described (30), except ml of loading solution containing pPVTK4 DNA (0-50 methanol was used as the fixative agent. Positive cells gg/ml) in a sterile 12 x 75 mm plastic round-bottom tube and exhibited bright nuclear fluorescence characteristic of the sonicated with three bursts of 0.5-sec duration at power large T antigen in SV40-infected cells. setting 1 of a Branson model 200 sonicator equipped with a two-step tapered microtip fitted with a plastic sleeve. This sonication loading method is an adaptation of a technique RESULTS developed for use with Dictyostelium discoideum (24, 29). Trial experiments indicated that the viability of LTK- fibro- Use of Scrape Loading and Sonication Loading for Trans- blasts under these conditions is approximately 70%. The fection of Mammalian Cells. Scrape loading (23) and sonica- number of viable cells was determined by counting in the tion loading (24) have both been employed previously for presence of trypan blue. introduction of macromolecules such as fluorescently-la- One million viable cells loaded with pPVTK4 DNA either beled dextran and protein molecules into the cytoplasm of by scraping or by sonication were diluted into 10 ml of living eukaryotic cells. Previous use of the sonication loading complete culture medium supplemented with 100 ,M hypo- method has, however, been restricted to experiments with xanthine and 16 ,M thymidine (HT medium) in a 10-cm amoebae of the cellular slime mold D.