Short Technical Reports Macrophage Scavenger Receptor Confers an Adherent Phenotype to Cells in Culture BioTechniques 25:240-244 (August 1998)
ABSTRACT Several cell lines have become widely used in biotechnology, pharmaceutical and academic laboratories because of their desirable characteristics. Among these, the human embryonic kidney cell line HEK293 is one the most versatile and powerful for expression of recombinant proteins, permitting the establishment of stable cell lines in just three weeks. Unfortunately, HEK293 cells adhere weakly to tissue culture grade plastic. Therefore, without cumbersome and sometimes expensive modifications to equipment, these cells are not suitable for use in a number of extremely important applications, including robot-based, highthroughput drug screening formats, growth and expansion in roller bottles and expression cloning experiments. We have cloned and transfected the human Class A macrophage scavenger receptor into 293EBNA cells (293EM) and found that expression of this receptor confers a sufficiently adherent property to the cells as to render them usable with automated equipment. In addition, the 293EM cells are now able to adhere to the surface of roller bottles and to untreated glass substrates, allowing growth of these cells in formats for which they are not normally well suited.
pendent adhesion of macrophages to tissue culture-treated plastic (TCP; Reference 4). It is interesting to note that the adhesive properties of the MSR receptor were not transferrable by stable transfection into Chinese hamster ovary (CHO) cells (7). By contrast, stable transfection of MSR into human embryonic kidney (HEK)293 cells was successfully used by Cheung et al. (2) to confer an adherent phenotype to cells grown on TCP. The cell lines HEK293 and 293EBNA (293E, an isogenic derivative expressing Epstein-Barr virus [EBV] nuclear antigen 1 [EBNA1]) are normally able to adhere only weakly to TCP substrates. Native adherence is insufficient to render the cell lines useful in experiments involving rapid addition of reagents or washing by automated equipment, because the cells are rapidly lost under these conditions. Currently, one can achieve acceptable adherence of these lines to TCP by precoating the substrate with agents such as poly-D-lysine, fibronectin or rat tail collagen. Pre-coating is cumbersome, expensive and time-consuming. Expression of MSR in 293E cells permits immediate use of the cell line (i) in roller bottles, (ii) in expression cloning experiments that require cell growth on glass rather than plastic surfaces and (iii) with automated robots without any further preparations or modifications of the culture substrates.
MATERIALS AND METHODS
TTTAGGAC-3′ (beginning at position 34 relative to the start codon) and 5′GCATTATAAAGTGCAAGTGACTCC-3′ (spanning the stop codon and ending at position +1358). The resulting 1392-bp fragment was subcloned into expression vector pE3∆ to make construct-pE3∆/MSR8 (Figure 1). Generation and Identification of Adherent 293E Cell Lines Construct-pE3∆/MSR8 was co-transfected with the pPur Selection Vector (CLONTECH Laboratories, Palo Alto, CA, USA) into 293EBNA cells (Invitrogen, Carlsbad, CA, USA) using the calcium phosphate method described in Sambrook et al. (9), and cells were selected in the presence of Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5 mM glutamine, and 1 µg/mL puromycin for 2 weeks. Clonal isolates were tested for adherence in Falcon 96-well microplates (Becton Dickinson Labware, Bedford, MA, USA). Adherent cell lines were identified following experimental procedures (described in the legend to Figure 2) conducted on a Biomek 2000 Laboratory Automation Workstation (Beckman Instruments, Fullerton, CA, USA), or SkanWasher 300 (Skatron Instruments, Sterling, VA, USA). BSS (25 mM HEPES pH 7.5, 11.5 mM KCl, 115 mM NaCl, 0.6 mM MgSO4, 1.8 mM CaCl2, 0.2% bovine serum albumin [BSA], 5 mM dextrose) was used for the cell-washing procedure (SkanWasher) and for the reagent addition experiments (Biomek 2000).
Cloning the MSR Class A cDNA INTRODUCTION Macrophage scavenger receptors (MSRs) are trimeric integral membrane glycoproteins that are able to recognize a wide variety of polyanionic ligands including modified low-density lipoproteins (LDLs), bacterial endotoxins and lipopolysaccharides (1,3,7). The human MSR was first cloned by Matsumoto et al. (8). Physiologically, the receptor is thought to serve as a homing or retention molecule for macrophage localization (4,6). The receptor has also been shown to be responsible for the serum-dependent, divalent cation-inde240 BioTechniques
MSR Class A cDNA (hSR-A, formerly known as MSR Type I, GenBank Accession No. D90187) was amplified by reverse transcription polymerase chain reaction (RT-PCR) from human hypothalamic RNA. First-strand cDNA was generated by RT using a mixture of random hexamer primers and oligo(dT12-18) and the SUPERSCRIPT Choice System cDNA Synthesis Kit, as per manufacturer’s instructions (Life Technologies, Gaithersburg, MD, USA). PCR amplifications were carried out using oligonucleotide primers 5′-AATCAGTGCTGCTTTC-
Figure 1. Schematic map of construct-pE3∆ ∆/ MSR8. The vector contains the cytomegalovirus immediate early promoter (CMV pro), human MSR coding sequence, simian virus 40 (SV40) splice and poly(A) signals [SV40 IVS+p(A)], colE1 ori and ampicillin-resistance marker (amp).
Cells were visualized by fixing and staining with methylene blue as follows. Medium or buffer was removed from 96-well microplates by manual aspiration or by tapping plates upside down. Fifty microliters of a 2% methylene blue staining solution in 50% aqueous methanol were added to each well for 5 min, and the plate was immersed 3× in a large volume of tap water to remove excess dye. The plate was then allowed to dry, and the image of the stained cell monolayers was captured using NIH Image Version 1.61 public
domain software (ftp://zippy.nimh. nih.gov/pub/nih-image). Growth in Roller Bottles and on Glass Slides Roller bottles (850 cm2; Corning Costar, Cambridge, MA, USA) were seeded with 1.2 × 107 cells and rotated at 0.25 rpm. Observations were made daily for 10 days. Untreated glass slide Flaskettes (Nalge Nunc International, Naperville, IL, USA) were seeded with 105 EM4 or 293E cells. On day 4, slides containing the cells were subjected to a receptor binding assay as described in Shen et al. (10). Briefly, slides are washed 2× with phosphate-buffered saline (PBS) at room temperature. Cells are incubated with 1 mL of BSS for 1 h at room temperature, plastic hoods are removed from the Flaskettes and slides are dipped for 4 × 10 s in ice-cold PBS. Slides are tapped dry and stained with methylene blue for visualization.
RESULTS AND DISCUSSION Generation and Selection of 293E Lines Expressing MSR
Figure 2. 293E clonal cell lines expressing MSR. Between 5 × 103 to 2 × 104 cells for each cell line were plated in triplicate 40 h before the assay. Cells in row A were permanently fixed and stained with methylene blue before any experimental treatment. Cells in rows B and C were subjected to a mock high-throughput drug screening assay, which involved: (i) rinsing the monolayer 3× on the SkanWasher (350 µL added at 0.1 Barr pressure), (ii) addition of 50 µL BSS on a Biomek 2000 Workstation (at a rate of 200 µL/s), (iii) incubation for 60 min at 23°C, (iv) rinsing 5× on the SkanWasher, followed by (v) methylene blue staining.
Cell line 293E is permissive for episomal replication of plasmids that contain an EBV origin of replication (oriP) and has been shown to be an exceptionally powerful, versatile system for conducting expression cloning experiments (10) and constructing stably transfected cell lines (5). Plasmid pE3∆msr8 (Figure 1), which expresses the HSR-A coding sequence, was co-transfected with plasmid pPur into 293E cells, and 47 individual puromycin-resistant colonies were picked for clonal expansion. To identify which of the 47 clonally derived cell lines manifested an adherent phenotype, cells were plated in three adjacent wells in a 96-well microplate (Figure 2), and cultures were subjected to automated pipetting and washing procedures (see Materials and Methods). Figure 2 shows that untransfected 293E cells do not adhere during the test procedure. Clonally derived MSR expressing cell lines clearly exhibited a wide range of characteristics, from nonadherent (clones EM12 and
Short Technical Reports EM13) to partially adherent (clones EM6, EM7 and EM9–11) to very adherent (clone EM8) phenotypes. Behavior of MSR Expressing Cell Lines in High-Throughput Format
Growth and Adherent Characteristics on Glass Slides 293E cells have been used previously in expression cloning experiments
(10). These experiments require cells to be grown on glass slide Flaskettes for subsequent receptor-binding analysis. The glass slides must be pre-coated with an extracellular matrix (ECM)
Eleven out of forty-seven candidate cell lines exhibited more greatly adherent properties than the parental 293E line and were chosen for further testing. Parental 293E cells (poorly adherent) and CHO cells (highly adherent) were used to provide the benchmark against which adherent properties of the recombinant cell lines were measured. Cells were plated at increasing densities in half of the wells of a 96-well microplate, and two cell lines were tested per microplate (Figure 3). The parental 293E cells barely remained after the first washing step (column 5) and were completely removed by the end of the assay (columns 1–4). CHO cells remained as an undisrupted monolayer in all instances. Clone EM25 appears to be as adherent as CHO cells, while EM4 may bind slightly less tightly as evidenced by a faint ring in the center of the wells in columns 1–4. Since the monolayers of clones EM9 and EM11 were clearly disrupted, these lines were not used for further testing. Behavior of MSR Expressing Cell Lines in Roller Bottles Cells from EM4 and EM25 were used to seed roller bottles to further examine the ability of these lines to adhere to TCP. The parental 293E line was observed to form large clumps floating in the media with very few cells sticking to the walls of the roller bottle. By contrast, EM25 cells adhered well to the roller bottle and formed only occasional adherent clumps. Line EM4 adhered very well to the roller bottle surface and formed a single, crowded monolayer without clumps or balled cells (data not shown). The basis for these differences was not investigated further. Cells from EM4 remained attached in a monolayer to the plastic, even after weeks in a single roller bottle. We have expressed recombinant, secretable ligands for integrins in EM25 cells and have been able to produce 0.6–1 mg of 300-kDa protein per roller bottle per 10 days (data not shown). 242 BioTechniques
Figure 3. Test of 293MSR lines in 96-well, high-throughput screening format. Forty hours before assay, 104 cells/well were added to rows A and B, 2 × 104 cells/well were added to rows C and D and 4 × 104 cells/well were added to rows E and F. Cells in columns 6 and 7 were stained without treatment, cells in columns 5 and 8 were stained after washing the monolayer 3× on the SkanWasher and cells in columns 1–4 and 9–12 were subjected to washing, reagent addition and incubation conditions as described for Figure 2.
such as poly-D-lysine or fibronectin to ensure proper growth and adherence of 293E cells. We wished to investigate whether expression of MSR on 293E cells would obviate the need for precoating with ECM. As shown in Figure 4, EM4 cells grown on glass slides were not appreciably removed following a mock-receptor-binding assay (compare Figure 4, Panels C and D). By contrast, following the mock-receptor-binding assay, a significant proportion of the 293E cell population was lost from the slide (compare Figure 4, Panels A and B). EM4 cells were observed microscopically to grow with a flattened, highly adherent phenotype, whereas the 293E cells grew with a rounded phenotype, often forming clumps that adhered only weakly to the glass substrate (data not shown). Although we have not formally demonstrated that the adherent phenotype of our stably transfected 293E cells is due to expression of MSR (e.g., by use of neutralizing antibodies to antagonize the adherent property or by detection of MSR protein by Western blotting), we believe that this remains the most likely explanation. Two other possible explanations for the emergence of adherent 293E cells include spontaneous mutation or the preexistence of a subpopulation of adherent cells. The first possibility is unlikely because the frequency with which adherent clones were obtained (11 out of 47 clones) is several orders of magni-
tude higher than expected rates of spontaneous mutation. We believe the second possibility is also improbable because parental populations of 293E cells do not exhibit any obvious heterogeneous, adherent phenotype in our experiments. We do not observe any detectable percentage of untransfected cells that is able to adhere tightly to untreated plastic substrates. Furthermore, we observe that the stably transfected, adherent cell lines described above are significantly more resistant to trypsinization than parental 293E cells, requiring 2 min of treatment for elution from plastic vs. 20–30 s for untransfected parental cells. Finally, parental 293E cells can be quantitatively eluted from tissue culture grade plastic by a 30-s incubation in PBS supplemented with 2 mM EDTA, whereas EM4 and EM25 cells do not elute to any measurable degree in PBS-EDTA even after a 10-min incubation. The 293E cell line provides an extraordinary system for expression of recombinant proteins. Using episomal vectors in 293E cells, one can construct a stable cell line in just three weeks (5). The magnitude of expression of recombinant protein in the resulting cells is equal to, and often exceeds expression levels in the very best CHO line selected from hundreds of clonal isolates. Therefore, the construction of 293E cells, which express both EBNA1 and MSR, greatly expands the experimental repertoire of this line to include use in high-
Figure 4. Growth of EM4 cells on untreated glass slides. Cells were stained with methylene blue and photographed before (Panels A and C) or after (Panels B and D) a mock-receptor-binding assay. Panels A and B are 293E cells, whereas Panels C and D are EM4 cells.
Short Technical Reports throughput drug screening formats, growth in roller bottles and facilitated use in expression cloning experiments.
10.Shen, E.S., G.M. Cooke and R.A. Horlick. 1995. Improved expression cloning using reporter genes and EBV ori-containing vectors. Gene 156:235-239.
ACKNOWLEDGMENTS
Address correspondence to Dr. Robert A. Horlick, Pharmacopeia, Inc., 3000 Eastpark Blvd., Cranbury, NJ 08512, USA. Internet:
[email protected]
The authors wish to thank Dr. Ilana Stroke for the initial suggestion of using the MSR, and Dr. Stroke, Dr. Robert Swanson and Ms. Adriane Schilling for critical review of this manuscript.
REFERENCES 1.Ashkenas, J., M. Penman, E. Vasile, S. Acton, M. Freeman and M. Krieger. 1993. Structures and high and low affinity ligand binding properties of murine type I and type II macrophage scavenger receptors. J. Lipid Res. 34:983-1000. 2.Cheung, P.H., C.L. Webb, N. Aiyar, J. Disa, P.G. Lysko and G. Feuerstein (Eds.). 1997. Transient expression of CGRP receptor cDNA in genetically modified 293 cells; 1996 July 28–August 1; Montreal Canada. Peptide Receptors Symposium Proceedings; Abstract p. 1.12, p. 48. 3.Emi, M., H. Asaoka, A. Matsumoto, H. Itakura, Y. Kurihara, Y. Wada, H. Kanamori, Y. Yazaki et al. 1993. Structure, organization, and chromosomal mapping of the human macrophage scavenger receptor gene. J. Biol. Chem. 268:2120-2125. 4.Fraser, I., D. Hughes and S. Gordon. 1993. Divalent cation-independent macrophage adhesion inhibited by monoclonal antibody to murine scavenger receptor. Nature 364:343346. 5.Horlick, R.A., K. Sperle, L.A. Breth, C.C. Reid, E.S. Shen, A.K. Robbins, G.M. Cooke and B.L. Largent. 1997. Rapid generation of stable cell lines expressing corticotropin releasing hormone receptor for drug discovery. Prot. Expr. Purific. 9:301-308. 6.Hughes, D.A., I.P. Fraser and S. Gordon. 1995. Murine macrophage scavenger receptor: in vivo expression and function as receptor for macrophage adhesion in lymphoid and non-lymphoid organs. Eur. J. Immunol. 25:466-473. 7.Hughes, D.A., I.P. Fraser and S Gordon. 1994. Murine mPhi scavenger receptor: adhesion function and expression. Immunol. Lett. 43:7-14. 8.Matsumoto, A., M. Naito, H. Itakura, S. Ikemoto, H. Asaoka, I. Hayakawa, H. Kanamori, H. Aburatani et al. 1990. Human macrophage scavenger receptors: primary structure, expression, and localization in atherosclerotic lesions. Proc. Natl. Acad. Sci. USA 87:9133-9137. 9.Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. CSH Laboratory Press, Cold Spring Harbor, NY. 244 BioTechniques
Detection and Characterization of αβ-T-Cell Clonality by Denaturing Gradient Gel Electrophoresis (DGGE) BioTechniques 25:244-250 (August 1998)
Received 23 Februrary 1998; accepted 17 April 1998.
Alan K. Robbins and Robert A. Horlick Pharmacopeia Cranbury, NJ, USA
ABSTRACT Accumulation of T cells carrying identical T-cell receptors (TCR) is associated with a number of immunological and nonimmunological diseases. Therefore, it is of interest to be able to analyze complex T-cell populations for the presence of clonally expanded subpopulations. Here, we describe a simple method combining reverse transcription (RT)-PCR and denaturing gradient gel electrophoresis (DGGE) for rapid detection and characterization of T-cell clonality. The detection of clonally expanded T cells by DGGE relies on the fact that clonal transcripts have no junctional diversity and therefore resolve at a fixed position in the gel, which is determined by their melting properties. For polyclonal populations with a high degree of junctional diversity, the different DNA molecules will resolve at different positions in the gel and together will be revealed as a smear. For each of the TCR β-variable gene (BV)1–24 families, cloned transcripts were amplified and shown to resolve as distinct bands in the denaturing gradient gel, whereas the analysis of polyclonal T-cell populations resulted in a smear in the gel. The present method might prove useful to test for clonotypic T-cells in a variety of pathological and physiological conditions and for monitoring T-cell responses in diagnostic and therapeutic settings.
INTRODUCTION The T-cell receptor (TCR) is a clonally distributed heterodimer, usually consisting of an α-chain and a β-chain. The T-cell repertoire is generated by rearrangement of gene segments encoding the TCR chains in the germ line consisting of a variable (V), joining (J) and constant (C) part, and for the β-chain, consisting of diversity (D) parts also. During this process, additional diversity
