Minitransfection: a simple, fast technique for transfections

Journal of Virological Methods, 32 (1991) B-88 0 1991 Elsevier Science Publishers B.V.

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AD0h’/S0168851091001122 VIRMET 01137

Minitransfection: a simple, fast technique for transfections W-Z. Ho’, E. G6nczii12, A. Srinivasan*, S.D. Douglas’ and S.A. Plotkin’ ‘The Children’s Hospitalof Philadelphia, Divisionof InfectiousDiseases and Immunology.Philadelphia. Pennsylvania, USA. and ?he WistarlnstituteofAnatomy andBiology_Philadelphia, Pennsylvania, U.SA. (Accepted 3 I October 1990)

Summary A fast, simple and inexpensive minitransfection technique, using either a lipofection or a calcium phosphate coptecipitation method to introduce foreign DNA into living cells is presented. This technique is based on the use of 24-well or 96-well tissue culture plates and can be used for both transient and stable transfections. Because it is a microtechnique, only small amounts of DNA, cells and transfection reagents are necessary, and it is easy to handle multiple DNA transfections or cotransfections in different cell lines and in duplicates or triplicates. The technique can be used to study viral gene expression, virus replication and chloramphenicol acetyltransferase (CAT) assay in different cell lines, for example, in the large scale screening and testing of antiviral agents. Minitransfection;

Macrotransfection;

Lipofection

Introduction Advances in the molecular biology of viruses have been greatly facilitated by transfection techniques for introducing foreign DNA into living cells. Many methods have been developed (Celis, 1984). The most widely used is transfection mediated by either calcium phosphate, or DEAE-dextran (Graham and van der Eb, 1973; Sompayrat and Danna, 198 1). A lipofection technique, recently developed by Philip Felgner Correspondence too:W-Z. Ho, Division of Infectious Diseases and Immunology, The Children’s Hospital OfPhiladelphia. 34th and Civic Center Boulevard, Philadelphia, PA 19104, U.S.A.

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and coworkers (1987), also seems to be highly promising. However, all of these methods suffer from the disadvantages of being time-consuming, inconvenient or expensive, particularly when multiple transfections or cotransfections or multiple cell lines must be made. To overcome these problems, we have developed a simple, fast and inexpensive minitransfection method based on the use of 24-well or 96-well tissue culture plates. We have successfully used this technique for both transient and stable transfection in different cell lines, in studies of viral gene expression.

Materials and Methods Cell lines The human rhabdomyosarcoma (RD), neuroblastoma (SK-N-MC), and HeLa cell lines (obtained from the American Type Culture Collection, ATCC) were maintained as monolayercultures in modified Eagle’s medium supplemented with 7.5% fetal calf serum (FCS). A subline of human RD cells, stably transfected with the HIV-tat gene (RDtat), was established in our laboratory (Velpandi et al., 1990). Antibodies Monoclonal antibody (Mab) to human cytomegalovirus-immediate (HCMV-IE) was obtained from Chemicon International.

early antigen

Recombinant plasmids Construction of an HIV long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) plasmid (pLTR-CAT) and an HIV trans-activator gene-containing plasmid (pTAT) has been described previously (Rando et al., 1987). The recombinant plasmid pRL43a, which contains the HCMV-IE gene 1 and 2 (Pizzomo et al., 1988), and the plasmid pSV*-Neo (Southern and Berg, 1982) were also used. Mucrotransfection Calcium phosphate-mediated transfection Cells plated in 60-mm dishes one day previously (2 x lo6 RD, RDtat cells or 3 x 10” HeLa cells). They were transfected with cesium chloride purified DNA (10 l.tg/dish) using the calcium phosphate coprecipitation technique (Graham and van der Eb, 1973). Four to six hours later they were treated with 15% glycerol for 2 min at 4-6 h post-transfection and cultured for a further 48 h. For CAT assay the cells were finally harvested by 3 cycles of freezing and thawing technique in dry ice and 37°C water. Lipofectin-mediated transfection RD, RDtat and HeLa cells are plated in a 60-mm dish and cultured until they are 7O100% confluent. They were then transfected with the experimental DNA (10 l.tg/dish)

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using lipofectin (BRL) according to the protocols supplied by the manufacturer. Briefly, 30 pg of lipofectin and 10 pg of experimental DNA were added to the cells. After 14 h the lipofectin-DNA mixture was removed and cells were fed with fresh medium containing 10% fetal calf serum (FCS). They were cultured for an additional 34 h, and harvested for CAT assay by 3 cycles of freezing and thawing in dry ice and 37°C water. Minitransfection Minitransfection using lipofectin was performed in either a 24-well plate (Falcon 3047) or a 96-well U-bottomed plate (Nunc, l-63320) according to the following procedure. (1) Plate cells in each well of the 24-well or 96-well plate in culture medium containing 10% FCS. The number of cells seeded per well depended upon their size and growth rate. (2) Incubate the cells at 37°C in a CO* incubator until they are 70-100% confluent. (3) Immediately before transfection, remove the growth medium from each well and add 200 ml (24-well plate) or 50 ml (96-well plate) of Gpti-MEM I reduced serum ‘medium, (Gibco; Cat. No. 320-1985 BD). (4) To each well of a 24-well plate, add 0.1-3 pg of DNA and l-3 pg of lipofectin or to each well of a 96-well plate, add 0.02-l pg of DNA and 1 pg of lipofectin. Gently swirl the plate to mix lipofectin and DNA. (5) Incubate transfected cells for 5-24 h at 37°C in a COz incubator. The incubation time required for optimal transfection varies depending on the cell type and quality of DNA used. (6) Remove the lipofectin-DNA mixture and add to each well 1 ml (24-well plate) or 250 pl(96-well plate) fresh medium containing 10% FCS and incubate for an additional 2448 h. (7) To harvest the cells, quickly wash the cells once with water and then add 150 pl(24-well plate) or 50 p1(96-well plate) of water to each well. Incubate the lysed cells at room temperature for 10-15 min. (8) The cell lysate is transferred from a 24-well plate to a microcentrifuge tube, and centrifuged at 13000 rpm for 5 min. The cell lysate is centrifuged directly in its 96well plate at 1500 rpm for 5 min. The supematant in the microcentrifuge tube and each well of the 96-well plate is now ready for a CAT assay. To identify gene expression by immunofluorescent assay, RD or HeLa cells were seeded into a 24-well plate, each well of which contained a 12-mm round glass coverslip and 2 ml medium with 10% FCS. The minitransfection was performed in the same way as described above, except that at Step 7, the cells on the coverslip were washed once with PBS, fixed with cold acetone, and then stained with corresponding antibody. We have also used the calcium phosphate technique for minitransfection assay. In this case, we reduced the amounts of DNA and transfection reagents in proportion to the surface area of the cell culture supporters. Since the surface area of a 60-mm dish is about 10 times larger than each well of a 24-well plate, and 30 times larger than each well of a 96-well plate, we calculated a corresponding reduction of experimental DNA and reagents. Twenty-four to 48 h after transfection, the cells were harvested as described in Step 7 of the minitransfection. Stable transfection using minitransfection technique To transfect a neuroblastoma

cell line, SK-N-MC, stably with different HIV and

HCMV genes, SK-N-MC cells were seeded in each well of a 24-well plate and cultured until they were 60-70% confluent. One pg of each experimental DNA, 0.01 pg of plasmid containing neomycin gene and 1 pg of lipofectin were added to the cells, which were cultured for 14 h. After washing away the lipofectin-DNA, the cells were cultured for a further 34 h before trypsinizing and transferring to a 6-well plate. The cells were cultured in the 6-well plate for 2-3 days before being transferred to a 60mm dish in medium containing G4 18. After 5-7 days, most cells were dead, but colonies of cells containing both experimental DNA and the neomycin gene, or only the neomycin gene, were visible. These colonies were then tested for gene expression. CATactivity assay CAT activity was determined by using either i4C-labelled chloramphenicol (DuPont, Inc.) and thin layer chromatography, as described by Gorman et al. (1982), or by using “C-labelled butyryl CoA (DuPont) in the direct diffusion assay described by Neumann et al. (1987). In brief, for the latter technique, 50 ~1 of cell extract was mixed with 200 p.1of 125 mM Tris buffer (pH 7.8) containing 1.25 mM chloramphenicol in a liquid scintillation vial. To initiate the reaction, 4 p.1 of the “C-labelled butyryl CoA was added, and 5 ml of Econofluor were gently laid over the mixture. The vials were counted sequentially in a liquid scintillation counter for 0.1 min.

Results The comparison of the sensitivityof minitransfection and macrotransfection Three cell lines were used in the study, HeLa, RDtat and RD cells. RDtat cells produce HIV- 1 tat protein which can be identified by transfecting the cells with HIV-LTR linked to the CAT gene (Velpandi et al., 1990). RD is the parental cell line of RDtat, and can be efficiently transfected with HIV and HCMV constructs (Srinivasan et al., 1989). The sensitivity and specificity of the mini~~sfection using 24-well or 96-well plates was compared with macrotr~sfection using a 60-mm dish. Two ~ansfection techniques (lipofection and calcium phosphate) were employed in this study. When RD and HeLa cells were cotransfected with HIV-LTR-CAT and its transactivator, pTAT, similar results were observed using minitransfection and macrotransfection, as was also the case when RDtat cells were transfected with HIV-LTR-CAT alone (Fig. 1). Using calcium phosphate coprecipitation techniques, no differences in CAT activities between mini~ansfection and macrotr~sfection were seen in either HeLa or RD cells transfected with HIV-LTR-CAT alone or cotransfected with HIV-LTRCAT and pTAT (Fig. 2).

The efficiency of minitransfection was also checked by immuno~uorescent assay. RD cells in a 24-well plate or 60-mm dish containing 12-mm glass coverslips were

83 70

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=60 5 E

4o

8

30

520 B 0

10 0 60mm Dish

24~011 Plau

96well

Plate

Fig. 1. Comparison ofCAT activity driven by HIV-LTR in RD, RDtat and HeLa cell lines cultured in three different tissue cultme sm using the lipofection technique described in Materials and Methods. All transfections were performed in du@cate. The RDtat cells were transfected with 10 pg (60-mm dish) or 1 pg (24-well plate) or0.2 pg (%-well plate) of pLTR-CAT. The RD and HeLa cells were cotransfected with pLTR-CAT and 2 M (60-mm dish) or 0.2 erg (24-well plate) or 0.05 pg (%-well plate) of pTAT. The cells were harveatcd 2 days post-transfection and CAT activity was performed by the one-vial assay described in Materials and Methods.

lAC-

16 96 6&nmdlsh

16 24dpldb RD

91

6 66 6mnm&h

2

69

24wP@J HOk

Fig. 2. Calcium phosphate performed transfection with pLTR-CAT plus pTAT in RD and HeLa cells cultured in either 60-mm dishes or 24-well plates. The amount of pLTR-CAT or pTAT used in different culture supporters was the same as in the experiment shown in Fig. 1. Cells were harvested after 2 days and CAT activity was determined by using thin layer chromatography as described in Materials and Methods.

X6 TABLE

I

Minitransfection-mediated stable transfection in SK-N-MC

cells

Plasmid”

Gene

Micrograms added

pLTR-CAT pTAT pRL43a pRL53a

HIV- 1-LTR HIV- 1-TAT HCMV-IE,, IEI HCMV-IE, HCMV-IE2

2

pMF18

G4 I I-resistant colonie? _-__ 6

1

5

2 2 I

14 6 7

“An approximate ratio of the indicated plasmid to the neomycin containing plasmid for cotransfection is 50: I. ?he numbers indicate the colonies containing both pSV2-Neo and experimental plasmids which were identified as described in Results.

gene (pRL43a, pRL53a and pMP 18). Table 1 shows G418-resistant colonies resulting from cotransfections with pSV*-Neo and the plasmids containing HIV-l or HCMV genes. The colonies which contained HCMV-IE genes were identified by indirect immunofluorescence using the E 13 mouse monoclonal antibody directed against the major IE nuclear antigen (Ho et al., 1990). The presence of the HIV-LTR or HIV-tat genes was tested by the CAT assay.

Discussion

We have described the minitransfection method using either lipofectin or calcium phosphate for the introduction of foreign DNA into living cells and its optimal conditions in the cell lines tested in this study. This method offers a great advantage over the commonly used macrotransfection techniques because it is simpler, less expensive and more easily reproduced. Because the microtechnique operates on a smaller scale, cell numbers and the amounts of DNA, lipofectin and other reagents required for transfection are greatly reduced. It is particularly useful when only limited numbers of primary cells are available for transfection or when multiple DNA transfections must be carried out, (e.g. the selection of newly cloned, functional expression vectors, or testing for multiple cell lines). In addition, duplicates or triplicates for each transfected plasmid guarantee the accuracy and reproducibility of the results. We found that minitransfection is as sensitive as macrotransfection (Fig. 1). The lipofectin-DNA or calcium phosphate-DNA complexes might have more opportunity to contact cells because of the limited surface area in a 24-well or 96-well plate. RDtat or RD LTR-CAT cell lines (available in our lab) should provide convenient tools for studying the effect of drugs, antibodies or other factors on the function of HIV-l promoter or tat gene expression. Instead of using 3 to 5 cycles of freezing-thawing technique to harvest the cells, we used distilled water to lyse the cells according to the theory of osmosis. This modification greatly simplifies the cell harvesting step by eliminating the cumbersome scraping, transfer, wash, and freeze-thaw steps. The simplicity of the method allows

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for rapid and easy processing of multiple DNA transfections or cotransfections in several replicas. The water harvest technique is especially helpful when 96-well plates are used since it is otherwise difficult and time-consuming to scrape cells in 96-well plates. For CAT assays both 24-well and 96-well plates are feasible. However, 24well plates, in our experience, are more practical than 96-well plates for studying virus replication or gene expression by immunofluorescence. Both lipofectin and calcium phosphate could be used in the minitransfection technique. Presumably, DEAE-dextran could also be used. Lipofection, certainly, showed advantages over calcium phosphate with respect to simplicity, and higher transfection efficiency (Felgner et al., 1987). With the cell types we used, lipofection is more efficient than the calcium phosphate technique (data not shown). Lipofectin is very expensive, in particular when an experiment requires multiple DNA transfections or cotransfections, but minitransfection can overcome the problem of expense since only 1 pg of lipofectin (about $0.15) is required for each well of a 24-well plate. We also modified the procedure for transfection with lipofectin by adding the lipofectin and experimental DNA to the cells directly, instead of mixing them beforehand. This not only simplified the technique without sacrificing the transfection efficiency (data not shown), but also avoided the problem of lipofectin-DNA precipitation. The concentration of DNA used for an optimal CAT activity driven by HIV-LTR varies with cell type. In this study, the difference between the RDtat and HeLa might be due to the amount of HIV-tat protein produced in these cells. The RDtat cell line is a stably tat-transfected cell line, whereas HeLa cells were transfected with 2 pg of pTAT in a transient transfection. Transfection efficiency in different cell lines also plays a significant role in the CAT activity driven by HIV-LTR. We are able to obtain satisfactory results with only 1 pg of lipofectin in RDtat cells. Increasing the lipofectin concentration failed to increase the CAT activity. However, the lipofectin toxicity to the cells was observed when excessive lipofectin was applied for the transfection, which confirms the observation by Felgner et al. (1987). The toxicity also depends on the type of cell, the incubation time of exposure to lipofectinDNA mixture and the density of the cell culture. In summary, the quantity of DNA and lipofectin used, the cell number plated and the duration of lipofectin-DNA mixture incubation on cells all play important roles in minitransfection. These factors must be considered and pretested in order to obtain optimal results. Also, quantitative use of these factors is highly dependent upon the cell type and transfection techniques used.

Acknowledgements The authors are grateful to Dr Peter Andrews and Shirley Peterson for editorial help. This work was supported by grant AI 25822 from the National Institutes of Health.

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References Celis, J.E. (1984) Microinjection of somatic cells with micropipettes: comparison with other transfer techniques. Biochem. J. 223,281-291. Felgner, P.L., Gadek, T.R., Helm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, GM. and Danielsen, M. (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA 84,7413-7417. Gorman, C.M., Moffat, L.F. and Howard, B.H. (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2.1044-l 05 1. Graham, F.L. and van der Eb, A.J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52,456467. Ho, W.-Z., Harouse, J.M., Rando, R.F., Giinczol, E., Srinivasan, A. and Plotkin, S.A. (1990) Reciprocal enhancement of gene expression and viral replication between human cytomegalovirus and human immunodeficiency virus type 1. J. Gen. Virol. 71,97-103. Neumann, J.R., Morency, CA. and Russian, K.D. (1987) A novel rapid assay for chloramphenicol acetyltransferase gene expression. BioTechniques $444-447. Pizzomo, M.C., O’Hare, P., Sha, L., LaFemina, R.L. and Hayward, G.S. (1988) Transactivation and autoregulation of gene expression by the immediate-early region 2 gene products of human cytomegalovirus. J. Virol. 62,1167-l 179. Rando, R.F., Pellett, P.E., Luciw, P.A., Bohan, CA. and Srinivasan, A. (1987) Transactivations of human immunodeficiency virus by herpes viruses. Oncogene 1,13-l 8. Sompayrac, L.M. and Danna, K.J. (198 1) Efficient infection of monkey cells with DNA of simian virus 40. Proc. Natl. Acad. Sci. USA 78,7575-7578. Southern, P.J. and Berg, P. (1982) Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1,327-341. Srinivasan, A., York, D., Jannoun-Nasr R., Kalyanaraman, S., Swan, D., Benson, J., Bohan, C., Luciw, P.A., Schnoll, S., Robinson, R.A., DeSai, S.M. and Devare, S.G. (1989) Generations of hybrid human immunodeficiency virus by homologous recombinations. Proc. Natl. Acad. Sci. USA 86,6388-6392. Velpandi, A., Monken, C.E. and Srinivasan, A. (1990) Development of RD-tat cell lines: use in HIV recombination studies. J. Viral. Methods 29.291-302.