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Rapid determination of transgene copy number in stably-transfected mammalian cells by competitive PCR
J. Biochem. Biophys. Methods 40 (1999) 101–112
Rapid determination of transgene copy number in stably-transfected mammalian cells by competitive PCR Ping Fu, Paul Senior, Ross T. Fernley, Geoffrey W. Tregear, G. Peter Aldred* Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia
Abstract We describe here an application of the competitive PCR technique to the analysis of copy number of recombinant rat parathyroid hormone-related protein (rPTHrP) gene in stably-transfected murine erythroleukemia (MEL) cell lines. A single-copy reference gene (endogenous mouse PTHrP gene or mPTHrP) is used as an internal control. This control gene, present in the genome of MEL cells, shares the same primer binding sites as the rPTHrP cDNA but contains an internal PvuII site, which allows resolution of the amplified products after restriction enzyme digestion by polyacrylamide gel electrophoresis (PAGE). The transgene copy number is determined by the ratio of band intensity of the rPTHrP product to that of the mPTHrP product. Using this method, we have determined the copy number of the rPTHrP transgene from isolated genomic DNA, and compared the results with those obtained from Southern blot analysis. In addition, we have demonstrated that the procedure can be applied very simply to whole MEL cells without DNA extractions and that as few as 10 4 cells are required for the analysis. 1999 Elsevier Science B.V. All rights reserved. Keywords: Transgene copy number; Competitive PCR; Parathyroid hormone-related protein (PTHrP); Murine erythroleukemia (MEL) cells
1. Introduction Competitive PCR methods have been used widely in various areas for quantitation of *Corresponding author. Present address: School of Science, University of Ballarat, University Drive, Mt Helen, Victoria, 3350, Australia. Tel.: 161-3-5327-9243; fax: 161-3-5327-9240. 0165-022X / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0165-022X( 99 )00018-4
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cellular mRNA and amplification of oncogenes [1–3]. The technique is based on the addition of a known amount of competitor or external standard to PCR reaction tubes containing samples. Since the amplification of target gene and standard occurs in the same tube, variability due to reaction conditions, differences in sample preparation, primer efficiency, and tube effects is internally controlled. Any factors which may affect yields of PCR products will do so equally for both the test gene and the standard. The amount of target material can be determined from the ratio of amplification products derived from the test template to those arising from the competitor template. Two types of external standards have been used in competitive PCR quantitations, both of them having the same primer binding sites as the target gene. The first type of external standard [1–3] is identical to the genomic sequence of interest except for a small deletion, insertion, or new restriction site so that it can be easily distinguished by size from its genomic counterpart on gel electrophoresis. The second type of external standard [4] consists of an unrelated DNA flanked by identical PCR primer sites to those of the test gene. Although each of these approaches has certain advantages and limitations, minimising the difference in size and sequence between the standard and the target will ensure similar amplification properties of these two species [5]. PCR has been shown to be capable of amplifying templates directly from cells, sperm, or blood samples without DNA or RNA extraction [3,6,7]. More recently, PCR on whole cells has allowed one to detect a single target cell in a background of numerous non-target cells, and to evaluate the presence and number of a rare cell type found in human blood [8]. The technique is suitable for use with small numbers of cells, as it avoids the problem of template loss that can accompany sample transfers during DNA purification. Furthermore, the method allows quantitation of large numbers of samples in a short time. In 1992 Needham et al. [9] described a new mammalian expression system which utilises the human globin locus control region (LCR) to direct high-level expression of recombinant proteins in murine erythroleukemia (MEL) cells. Since the LCR has been shown to drive expression of heterologous genes in a position independent, gene copy-number dependent manner [10], the level of heterologous protein expression directly corresponds to the copy number of the transgene in MEL cell genome [9]. In our study of the expression of rat PTHrP [11] in the LCR / MEL cell system, it is necessary to isolate stably-transfected cell clones which contain high copy numbers of the transgene in order to maximise protein production. Previously, this analysis was performed by Southern blot [9,12] which requires multiple manipulations and transfers. Also, it is time-consuming, costly and subject to variability depending on the characteristics of the probe used. In addition, resources necessary for DNA extraction and gel electrophoresis can become prohibitively expensive when working with large numbers of samples. This prompted us to develop a protocol that allows for the rapid and efficient quantitation of copy number of the recombinant gene. In this paper, we describe a novel application of the competitive PCR technique for the determination of rPTHrP transgene copy number in stably-transfected MEL cells. The method utilises a single-copy endogenous mouse PTHrP gene [13] as an internal standard to monitor the rate and efficiency of PCR amplification. As the standard gene is present in the mouse genome, errors associated with the addition of external standard
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can be eliminated. In addition, the method is applicable to samples of a very small size (as few as 10 4 cells).
2. Materials and methods
2.1. Materials Restriction enzyme PvuII, Klenow fragment, RNase A and Taq DNA polymerase were purchased from Promega Ltd. The radiochemicals [a- 32 P]dATP and [a- 32 P]dCTP (both 3000 Ci / mmol) were obtained from Bresatec Ltd. Geneticin (G 418 ) and glutamine were purchased from Gibco Inc. DMEM culture medium and penicillin / streptomycin were purchased from Trace Biosciences Ltd. Fetal calf serum was purchased from CSL Diagnostic. The plasmid PLP m10 containing the cDNA of rat PTHrP was a gift from Dr D. Goltzman, McGill University, Canada [11]. Plasmids of the pEC3 and pGSE1417, originally constructed by Needham et al. [9], were gifts from Prof. W.J. Brammar, Leicester University, UK. MEL cells (F4N strain) were kindly provided by Dr M. Hibbs at the Ludwig Institute, Australia. Oligonucleotide primers were synthesised using an Applied Biosystems DNA synthesiser model 380A. The sequences are as follows: sense primer: 59-AACTCCAAACCTGCTCCCAACACC-39; antisense primer: 59-TTA / GAGCTGGGCTCCAGC /GGAGGT-39.
2.2. rPTHrP expression constructor A HindIII site and a BamHI site were introduced to the rPTHrP cDNA at each end by PCR on the plasmid PLP m10 [11]. The PCR product was cloned into the HindIII / BamHI site of the pEC3 vector. The final expression vector was generated by subcloning the expression cassette from the pEC3 / rPTHrP into the human globin LCR plasmid pGSE1417 to generate the pLCR / EC3 / rPTHrP expression cassette [9].
2.3. MEL cell transfection and DNA preparation The expression construct was linearised by PvuI digestion and transfected into MEL cells by electroporation [14]. Stably-transfected MEL cells were cultured in DMEM supplemented with 2 mM glutamine, 10% fetal calf serum, 50 IU / ml of penicillin, 50 mg / ml of streptomycin and 800 mg / ml of G418 until nearly confluent (3–4310 6 cells / ml). Genomic DNA was prepared according to the method of Antoniou [14]. Ten ml of cell suspension were pelleted in a bench-top centrifuge and washed with 5 ml of PBS. The cells were then resuspended in 4 ml of cell lysis buffer (50 mM Tris–HCl, pH 7.5; 100 mM NaCl; 5 mM EDTA) containing 1% SDS and 0.5 mg / ml proteinase K, and incubated at 378C overnight. The cell lysis mixture was extracted with an equal volume of phenol–chloroform, and genomic DNA was precipitated with 2.5 volumes of ethanol. The DNA was dissolved in 3 ml of TE (10 mM Tris–HCl, pH 7.5; 1 mM EDTA) containing 10 mg / ml DNase-free RNase A, and incubated at 378C for 15 min. The DNA
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was again extracted with phenol–chloroform, and reprecipitated with 0.2 M sodium acetate and 2.5 volumes of ethanol. The genomic DNA was then dissolved in 0.5 ml of water and stored at 48C.
2.4. Southern analysis The 0.59 kb rPTHrP cDNA probe for Southern blot analysis was labelled with [a- 32 P]dCTP using the random priming method [15]. Genomic DNA samples isolated from individual clones of rPTHrP stably-transfected MEL cells were digested with BamHI and HindIII restriction enzymes. The digested DNA samples were electrophoresed on a 0.8% agarose gel and transferred onto a Hybond N 1 filter in the presence of 0.5 N NaOH, 1.5 M NaCl. The filter was then neutralised in Tris buffer (1.5 M NaCl, 0.5 M Tris–HCl, pH 7.2) and baked at 808C for 2 h. The hybridisation was performed in 53 SSC, 53 Denhardt’s, 1% SDS and 20 mg / ml denatured herring sperm DNA at 658C overnight. The filter was washed four times in 0.13 SSC, 0.1% SDS at 608C, and exposed to an imaging plate. Bands were quantitated using the FUJIX Bio-image Analyser BAS2000 (Fuji, Photo Film Co. Ltd.).
2.5. PCR analysis 2.5.1. PCR on DNA The standard PCR reaction mixture contained 0.5 mg of genomic DNA, 0.8 mM of each primer, 100 mM of each dNTP (dATP, dGTP, dTTP, dCTP), 0.3 ml of [a- 32 P]dATP, 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 and 1.5 units of Taq DNA polymerase. All competitive PCR reactions were performed in a total volume of 50 ml and cycled in a DNA thermal cycler 4800 (Perkin-Elmer Cetus) with an overlay of mineral oil. For each set of PCR reactions, master solutions were prepared so that all reaction tubes contained the same reaction mix. The thermal cycle conditions were: 948C for 3 min (initialising step), 948C for 1 min, 608C for 1 min, 728C for 1 min (repeated for 25 cycles) and 728C for 10 min (final extension). 2.5.2. PCR on cells MEL cells were spun down and washed with TE (10 mM Tris–HCl, pH 8.3, 1 mM EDTA). Cells were resuspended in 10 ml of TE and lysed by heating to 958C for 5 min. Proteinase K was added to a concentration of 200 mg / ml. Samples were incubated at 568C for 1 h, then at 958C for 10 min to inactivate proteinase K. The entire reaction mixture from the previous step was amplified using the conditions described above, except that the MgCl 2 concentration was reduced to 1 mM. After the PCR amplification, a 15-ml aliquot of each reaction was digested with 5 units of PvuII restriction enzyme in a 30-ml volume at 378C for 2 h. Ten ml of each digested sample were electrophoresed on a 6% polyacrylamide gel in 23 TBE buffer. The gel was then dried under vacuum, and exposed to an imaging plate for 1–2 h. The imaging plate was scanned, and the data were analysed using the BAS2000.
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3. Results and discussion
3.1. Strategy of the determination of transgene copy number The procedure described for the determination of foreign gene copy number is illustrated in Fig. 1. PCR primers were designed to hybridise to regions of high sequence conservation between the rat PTHrP and mouse PTHrP genes [11,13]. The 59 and 39 primers are located at amino acids 45–52 and 130–137 of the mouse PTHrP gene exon 4 (amino acids 45–52 and 132–139 in the rat PTHrP gene) respectively. The 59 primer sequence is 100% identical for both PTHrP genes. Due to the presence of two nucleotide
Fig. 1. Procedure for determining foreign gene copy number in stably-transfected cells. The process is illustrated using murine erythroleukemia cells transformed with pLCR / EC3 / rPTHrP expression cassette. Individual steps are described in the text. * Represents a correction factor resulting from differences in dA contents of the two PCR products.
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differences in the positions of the 39 primer regions of two genes, degenerate primers comprising a mixture of two nucleotide sequences were used in order to achieve equal amplification. Genomic DNA isolated from stably-transformed MEL cells was subjected to PCR amplification in the presence of [a- 32 P]dATP. The addition of trace amounts of radioactive nucleotide allowed for the direct quantitation of PCR products after gel separation, without affecting the reaction kinetics. The amplification led to the production of a 277-bp product from the endogenous mouse PTHrP gene and a 283-bp product from the rat PTHrP transgene. The mPTHrP product contains a cleavage site for the restriction enzyme PvuII, which is absent in the rPTHrP product. Cleavage of this mouse product with PvuII yields a major fragment of 211 bp and a smaller fragment of 66 bp. Intact and digested PCR products were then separated by PAGE and the individual bands were quantitated. The recombinant gene copy number can be obtained by calculating the ratio of 283-bp and 211-bp bands (see Fig. 1). The 66-bp digested fragment of the mPTHrP product was not measured since the results would have been affected by co-migration of this fragment with other low molecular weight radioactive products, such as primer dimers. This rapid method for quantitating the copy number of a recombinant gene in stably-transfected mammalian cells can be easily applied to any transgene of known sequence. The technique utilises the single-copy endogenous gene as an internal reference standard. As the standard gene is endogenous to the genome, errors arising from the addition of external standard and quantitation of standard or sample can be avoided. We have applied the method to the determination of copy number of rat PTHrP transgene in stably-transfected mouse erythroleukemia cells.
3.2. Heteroduplex formation affected the PCR assay The initial PCR amplification was carried out with 0.3 mM of each primer and 35–40 cycles. Under the conditions used, the intact PCR products (283 bp and 277 bp) showed similar mobility on the gel electrophoresis (Fig. 2A, lane 2, band b). However, an additional band showing a slower mobility than the expected band was observed (Fig. 2A, lane 2, band a). This band remained unchanged after PvuII digestion (Fig. 2A, lane 1, band a), indicating that it has no PvuII site. To investigate whether this slow moving band was a non-specific product or a heteroduplex DNA generated by cross-hybridisation of the mPTHrP and rPTHrP products, 5 ml of PCR products were denatured by heating at 1008C for 3 min before loading on the same gel. With this treatment, both the target band and slow moving band were found to have shifted to the same position corresponding to that of a single-stranded DNA (Fig. 2A, lane 3, band d). The result strongly suggests that the slow moving band is a heteroduplex DNA. Fig. 2B shows that the heteroduplex product increased from 5% to 40% of total PCR products when PCR cycles increased from 25 to 40. Optimisation of PCR amplification conditions was carried out to reduce the formation of heteroduplex DNA. The optimum amplification conditions were achieved by increasing the concentration of PCR primers to 0.8 mM and decreasing the PCR cycle number to 25. The formation of heteroduplex DNA during the competitive PCR amplification is a factor which is likely to introduce errors into the assay. Heteroduplex DNA is resistant
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Fig. 2. Heteroduplex formation during the competitive PCR amplification of genomic DNA isolated from stably-transfected MEL cells. (A) Identification of heteroduplex product after 40 cycles of amplification. Lane 1, PCR products digested by PvuII; lane 2, PCR products; lane 3, PCR products preheated to 1008C for 3 min before being loaded on the gel. (a) Heteroduplex band; (b) 277 bp / 283 bp band resulting from amplification of the mouse and rat PTHrP genes; (c) 211 bp band from the mouse PTHrP product after the PvuII digestion; (d) single-stranded DNA band. (B) The effect of number of PCR cycles on heteroduplex formation: lane 1, 25 cycles; lane 2, 28 cycles; lane 3, 31 cycles; lane 4, 34 cycles; lane 5, 37 cycles; lane 6, 40 cycles; lane 7, negative control (no DNA template).
to restriction enzyme digestion, and therefore it is not easily distinguished from the target and competitor. The effect of heterologous duplex formation on electrophoretic mobility and the generation of heteroduplex DNA during PCR amplification have been reported previously [16,17]. According to Ruano and Kidd [17], in later PCR cycles, during which the high concentrations of PCR products are present, cross-hybridisation between products of the competitor and target gene can efficiently compete with the hybridisation of PCR primers to templates, resulting in the formation of DNA heterodimer. As the heteroduplex formed in the last PCR cycle is not denatured, it can form a significant proportion of the final products. Jenson and co-worker [18] reported a similar finding to that described here, indicating that the formation of heteroduplex DNA could be decreased by increasing PCR primer concentrations and / or by decreasing PCR cycles. Under these conditions where a high primer / template ratio is present, the formation of primer / template duplex is kinetically favoured over the rehybridisation of previously synthesised products, so that the formation of heteroduplex can be reduced.
3.3. PCR and Southern blot analyses on genomic DNA Fig. 3 illustrates the result of a PCR amplification on genomic DNA isolated from stably-transfected MEL cells and control MEL cells. In products amplified from DNA of
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Fig. 3. Polyacrylamide gel electrophoresis of co-amplified recombinant rat PTHrP gene product (283 bp) and endogenous mouse PTHrP gene product (211 bp) after 25 PCR cycles and PvuII digestion. M, markers; PCR products from MEL cells transfected with pLCR / EC3 / rPTHrP single cell clones (lanes 1–7); PCR products from MEL cells transfected with pLCR / EC3 / rPTHrP pooled populations (lanes 8–10); PCR products from control MEL cells (lane 11); negative control (no DNA template, lane 12). The rPTHrP transgene copy number: lane 1, 2; lane 2, 9; lane 3, 2; lane 4, 5; lane 5, 1; lane 6, 2; lane 7, 2; lane 8, 5; lane 9, 3; lane 10, 6; lane 11, 0.
transformed MEL cells, two bands were observed after PvuII digestion, representing the 283 bp rPTHrP product and the 211 bp mPTHrP product (lanes 1–10). In contrast, only the 211 bp mPTHrP product was observed with the DNA isolated from control MEL cells (lane 11). The latter sample also serves as a control of the PvuII digestion in our PCR assay. To test whether the PCR amplification result reflected the recombinant gene copy number present in MEL cell lines, we performed both the PCR assay and Southern blot analysis on the same DNA samples. For Southern blot analysis, the DNA samples were digested with restriction enzymes HindIII and BamHI. A Southern blot of the DNA gel was hybridised with a rat PTHrP cDNA probe. In the control sample, the probe only hybridised to the 5.1 kb fragment of mPTHrP [13], while in samples isolated from stably-transfected cells, the probe hybridised to the 0.59 kb rPTHrP fragment as well as to the 5.1 kb mPTHrP fragment. The intensity of each band was quantitated, and copy numbers of the transgenes were calculated. Table 1 shows a comparison of results of the transgene copy number determined by the competitive PCR and Southern blot analyses. Results from two different methods are consistent, indicating the reliability of PCR method for the determination of copy number of the transgene.
3.4. PCR analysis on MEL cells In several previous reports, PCR amplification has been successful using whole cells as the starting material rather than extracted DNA [3,6–8]. Hence we investigated the possibility of performing our PCR assay directly on lysed MEL cells. A range of dilutions of rPTHrP stably-transfected MEL cells was tested (from 5310 2 to 5310 5 cells per reaction). Cells were lysed by heating to 958C for 5 min, followed by a proteinase K digestion to remove contaminating proteins. Fig. 4 shows PCR products
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Table 1 Comparison of the Southern blot and PCR analyses for the determination of the rPTHrP transgene copies in MEL cells a Genomic DNA samples
Southern blot analysis
PCR assay
1 2 3 4 5 6 7 8
4.4 20.0 4.0 0.8 3.2 3.4 10.8 8.0
4.6 18.8 4.0 1.4 4.4 5.6 10.8 7.0
a DNA samples 1–6 were prepared from MEL cells transfected with pLCR / EC3 / rPTHrP single cell clones. DNA samples 7–8 were obtained from MEL cells transfected with pLCR / EC3 / rPTHrP pooled populations.
Fig. 4. Competitive PCR performed directly on whole MEL cell lysates without DNA extraction. The cells contain an average of four copies of the rPTHrP transgene. (A) Amplified PCR products. (B) PCR products digested by the PvuII restriction enzyme. Numbers of MEL cells used as templates for the reaction were as follows: lane 1, 5310 2 ; lane 2, 1310 3 ; lane 3, 5310 3 ; lane 4, 1310 4 ; lane 5, 5310 4 ; lane 6, 1310 5 ; lane 7, 5310 5 ; lane 8, negative control (no cells).
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obtained from amplification of the rat and mouse PTHrP genes from whole MEL cell lysates. Results indicated that 10 4 cells provided sufficient template to yield a positive signal after 25 amplification cycles and could be reliably used for transgene copy number determination. The sensitivity could be further increased 10-fold (i.e. 10 3 cells) if nested PCR was performed using a second pair of PCR primers (data not shown). The ability to conduct competitive PCR analysis on whole MEL cells is one of the major factors contributing to the reduction in processing time. It is important to note, however, that this method is found to be successful only if contaminating proteins are removed prior to PCR amplification by proteinase K digestion. The presence of large amounts of contaminating proteins not only reduces the efficiency of PCR amplification, but also inhibits electrophoretic mobility of the PCR products. Thus the proteinase K digestion is essential and should be conducted routinely prior to PCR amplification. The major advantage of this technique of competitive PCR over Southern blotting for determining transgene copy number is the speed with which large numbers of samples can be processed. MEL cells, like many other mammalian cell types, have a very slow growth rate, with cell divisions occurring only every 10–12 h. Screening of recombinant cell line clones by Southern blot requires at least 2 weeks for expansion of the cell line from a single cell to 10 7 cells. Subsequent DNA extraction and Southern blot analysis requires another 3–4 days. In contrast, using this competitive PCR method, PCR analysis of a cell line expanded from a single cell can take place after only 1 week. The analysis itself (including PCR amplification, restriction enzyme digestion, gel electrophoresis and band quantitation) can be completed within a day. If necessary, the process could be further streamlined by, for example, increasing the concentration of the radioactive nucleotide or utilising a more rapid thermal cycler. Thus, by screening recombinant cell lines with competitive PCR, time consuming and / or costly procedures, such as tissue culture, DNA extraction, Southern blot transfer, probe preparation and blot hybridisation, can be avoided.
4. Simplified description of the method and its (future) applications We describe here a rapid method for quantitating the copy number of a recombinant gene in stably-transfected mammalian cells that can be easily applied to any transgene of known sequence. The novel aspect of the technique is the use of the single-copy endogenous gene as an internal reference standard. The copy number is then determined by the ratio of the transgene to the endogenous gene. The transgene is differentiated from the endogenous gene by restriction enzyme cleavage of the endogenous gene. However, it should be noted that the method is only applicable when two highly homologous regions are found within the transgene and endogenous gene. In addition, we have demonstrated that the technique can be used directly on MEL cells without DNA extraction, and that a PCR of 25 cycles provides adequate signal sensitivity when 10 4 MEL cells are used. These conditions are also optimised to reduce heteroduplex formation. Since the use of mammalian expression systems to produce recombinant proteins or as a means of studying gene function is becoming more common, the ability to determine
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transgene copy number easily and rapidly will prove extremely valuable. In addition to its use in transfected cells for protein expression, this method may be applicable to measuring copy number in transgenic animals and plants.
Acknowledgements We thank Drs P. Roche and J. Gunnersen for their helpful discussions. We also thank Dr E.H. Kachab and S. Khoury for the synthesis of oligonucleotides. This work was supported by an institute grant to the Howard Florey Institute from the National Health and Medical Research Council of Australia.
References [1] Gilliland G, Perrin S, Blanchard K, Bunn HF. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci USA 1990;87:2725–9. [2] Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA 1989;86:9717–21. [3] Harlow SP, Stewart CC. Quantitation of c-myc gene amplification by a competitive PCR assay system. PCR Methods Appl 1993;3:163–8. [4] Uberla K, Platzer C, Diamantstein T, Blankenstein T. Generation of competitor DNA fragments for quantitative PCR. PCR Methods Appl 1991;1:136–9. [5] McCullock RK, Choong CS, Hurley DM. An evaluation of competitor type and size for use in the determination of mRNA by competitive PCR. PCR Methods Appl 1995;4:219–26. [6] Kawasaki ES. Sample preparation from blood, cells, and other fluids. In: Innis MA, Gelfan DH, Sninsky JJ, White TJ, editors, PCR protocols: a guide to methods and applications, San Diego, CA: Academic Press, 1990, pp. 146–52. [7] Li H, Gyllensten UB, Cui X, Saiki RK, Erilich HA, Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 1988;335:414–7. [8] Molesh DA, Hall JM. Quantitative analysis of CD34 1 stem cells using RT-PCR on whole cells. PCR Methods Appl 1994;3:278–84. [9] Needham M, Gooding C, Hudson K, Antoniou M, Grosveld F, Hollis M. LCR / MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells. Nucl Acids Res 1992;20:997–1003. [10] Talbot D, Collis P, Antoniou M, Vidal M, Grosveld F, Greaves DR. A dominant control region from the human beta-globin locus conferring integration site-independent gene expression. Nature 1989;338:352– 5. [11] Yasuda T, Banville D, Rabbani SA, Hendy G, Goltzman D. Rat parathyroid hormone-like peptide: comparison with the human homologue and expression in malignant and normal tissue. Mol Endocrinol 1989;3:518–25. [12] Fourney RM, Aubin R, Dietrich KD, Paterson MC. Determination of foreign gene copy number in stably transfected cell lines by Southern transfer analysis. In: Murray EJ, editor, Methods in molecular biology: gene transfer and expression protocols, Vol. 7, Clifton, NJ: The Human Press, 1991, pp. 381–95. [13] Mangin M, Ikeda K, Broadus AE. Structure of the mouse gene encoding parathyroid hormone-related peptide. Gene 1991;95:195–202. [14] Antoniou M. Induction of erythroid-specific expression in murine erythroleukemia (MEL) cell lines. In: Murray EJ, editor, Methods in molecular biology: gene transfer and expression protocols, Vol. 7, Clifton, NJ: The Human Press, 1991, pp. 421–34. [15] Sambrook J, Fritsch EF, Maniatis T. 2nd ed, Molecular cloning: a laboratory manual, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1989.
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[16] Hsiech CH, Griffith JD. Deletions of bases in one strand of duplex DNA, in contrast to single-base mismatches, produce highly kinked molecules: possible relevance to the folding of single-stranded nucleic acids. Proc Natl Acad Sci USA 1989;86:4833–7. [17] Ruano G, Kidd KK. Modeling of heteroduplex formation during PCR from mixtures of DNA templates. PCR Methods Appl 1992;2:112–6. [18] Jenson MA, Strano N. Effect of PCR conditions on the formation of heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacer regions. PCR Methods Appl 1993;3:186–94.
Rapid determination of transgene copy number in stably-transfected mammalian cells by competitive PCR Ping Fu, Paul Senior, Ross T. Fernley, Geoffrey W. Tregear, G. Peter Aldred* Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia
Abstract We describe here an application of the competitive PCR technique to the analysis of copy number of recombinant rat parathyroid hormone-related protein (rPTHrP) gene in stably-transfected murine erythroleukemia (MEL) cell lines. A single-copy reference gene (endogenous mouse PTHrP gene or mPTHrP) is used as an internal control. This control gene, present in the genome of MEL cells, shares the same primer binding sites as the rPTHrP cDNA but contains an internal PvuII site, which allows resolution of the amplified products after restriction enzyme digestion by polyacrylamide gel electrophoresis (PAGE). The transgene copy number is determined by the ratio of band intensity of the rPTHrP product to that of the mPTHrP product. Using this method, we have determined the copy number of the rPTHrP transgene from isolated genomic DNA, and compared the results with those obtained from Southern blot analysis. In addition, we have demonstrated that the procedure can be applied very simply to whole MEL cells without DNA extractions and that as few as 10 4 cells are required for the analysis. 1999 Elsevier Science B.V. All rights reserved. Keywords: Transgene copy number; Competitive PCR; Parathyroid hormone-related protein (PTHrP); Murine erythroleukemia (MEL) cells
1. Introduction Competitive PCR methods have been used widely in various areas for quantitation of *Corresponding author. Present address: School of Science, University of Ballarat, University Drive, Mt Helen, Victoria, 3350, Australia. Tel.: 161-3-5327-9243; fax: 161-3-5327-9240. 0165-022X / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0165-022X( 99 )00018-4
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cellular mRNA and amplification of oncogenes [1–3]. The technique is based on the addition of a known amount of competitor or external standard to PCR reaction tubes containing samples. Since the amplification of target gene and standard occurs in the same tube, variability due to reaction conditions, differences in sample preparation, primer efficiency, and tube effects is internally controlled. Any factors which may affect yields of PCR products will do so equally for both the test gene and the standard. The amount of target material can be determined from the ratio of amplification products derived from the test template to those arising from the competitor template. Two types of external standards have been used in competitive PCR quantitations, both of them having the same primer binding sites as the target gene. The first type of external standard [1–3] is identical to the genomic sequence of interest except for a small deletion, insertion, or new restriction site so that it can be easily distinguished by size from its genomic counterpart on gel electrophoresis. The second type of external standard [4] consists of an unrelated DNA flanked by identical PCR primer sites to those of the test gene. Although each of these approaches has certain advantages and limitations, minimising the difference in size and sequence between the standard and the target will ensure similar amplification properties of these two species [5]. PCR has been shown to be capable of amplifying templates directly from cells, sperm, or blood samples without DNA or RNA extraction [3,6,7]. More recently, PCR on whole cells has allowed one to detect a single target cell in a background of numerous non-target cells, and to evaluate the presence and number of a rare cell type found in human blood [8]. The technique is suitable for use with small numbers of cells, as it avoids the problem of template loss that can accompany sample transfers during DNA purification. Furthermore, the method allows quantitation of large numbers of samples in a short time. In 1992 Needham et al. [9] described a new mammalian expression system which utilises the human globin locus control region (LCR) to direct high-level expression of recombinant proteins in murine erythroleukemia (MEL) cells. Since the LCR has been shown to drive expression of heterologous genes in a position independent, gene copy-number dependent manner [10], the level of heterologous protein expression directly corresponds to the copy number of the transgene in MEL cell genome [9]. In our study of the expression of rat PTHrP [11] in the LCR / MEL cell system, it is necessary to isolate stably-transfected cell clones which contain high copy numbers of the transgene in order to maximise protein production. Previously, this analysis was performed by Southern blot [9,12] which requires multiple manipulations and transfers. Also, it is time-consuming, costly and subject to variability depending on the characteristics of the probe used. In addition, resources necessary for DNA extraction and gel electrophoresis can become prohibitively expensive when working with large numbers of samples. This prompted us to develop a protocol that allows for the rapid and efficient quantitation of copy number of the recombinant gene. In this paper, we describe a novel application of the competitive PCR technique for the determination of rPTHrP transgene copy number in stably-transfected MEL cells. The method utilises a single-copy endogenous mouse PTHrP gene [13] as an internal standard to monitor the rate and efficiency of PCR amplification. As the standard gene is present in the mouse genome, errors associated with the addition of external standard
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can be eliminated. In addition, the method is applicable to samples of a very small size (as few as 10 4 cells).
2. Materials and methods
2.1. Materials Restriction enzyme PvuII, Klenow fragment, RNase A and Taq DNA polymerase were purchased from Promega Ltd. The radiochemicals [a- 32 P]dATP and [a- 32 P]dCTP (both 3000 Ci / mmol) were obtained from Bresatec Ltd. Geneticin (G 418 ) and glutamine were purchased from Gibco Inc. DMEM culture medium and penicillin / streptomycin were purchased from Trace Biosciences Ltd. Fetal calf serum was purchased from CSL Diagnostic. The plasmid PLP m10 containing the cDNA of rat PTHrP was a gift from Dr D. Goltzman, McGill University, Canada [11]. Plasmids of the pEC3 and pGSE1417, originally constructed by Needham et al. [9], were gifts from Prof. W.J. Brammar, Leicester University, UK. MEL cells (F4N strain) were kindly provided by Dr M. Hibbs at the Ludwig Institute, Australia. Oligonucleotide primers were synthesised using an Applied Biosystems DNA synthesiser model 380A. The sequences are as follows: sense primer: 59-AACTCCAAACCTGCTCCCAACACC-39; antisense primer: 59-TTA / GAGCTGGGCTCCAGC /GGAGGT-39.
2.2. rPTHrP expression constructor A HindIII site and a BamHI site were introduced to the rPTHrP cDNA at each end by PCR on the plasmid PLP m10 [11]. The PCR product was cloned into the HindIII / BamHI site of the pEC3 vector. The final expression vector was generated by subcloning the expression cassette from the pEC3 / rPTHrP into the human globin LCR plasmid pGSE1417 to generate the pLCR / EC3 / rPTHrP expression cassette [9].
2.3. MEL cell transfection and DNA preparation The expression construct was linearised by PvuI digestion and transfected into MEL cells by electroporation [14]. Stably-transfected MEL cells were cultured in DMEM supplemented with 2 mM glutamine, 10% fetal calf serum, 50 IU / ml of penicillin, 50 mg / ml of streptomycin and 800 mg / ml of G418 until nearly confluent (3–4310 6 cells / ml). Genomic DNA was prepared according to the method of Antoniou [14]. Ten ml of cell suspension were pelleted in a bench-top centrifuge and washed with 5 ml of PBS. The cells were then resuspended in 4 ml of cell lysis buffer (50 mM Tris–HCl, pH 7.5; 100 mM NaCl; 5 mM EDTA) containing 1% SDS and 0.5 mg / ml proteinase K, and incubated at 378C overnight. The cell lysis mixture was extracted with an equal volume of phenol–chloroform, and genomic DNA was precipitated with 2.5 volumes of ethanol. The DNA was dissolved in 3 ml of TE (10 mM Tris–HCl, pH 7.5; 1 mM EDTA) containing 10 mg / ml DNase-free RNase A, and incubated at 378C for 15 min. The DNA
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was again extracted with phenol–chloroform, and reprecipitated with 0.2 M sodium acetate and 2.5 volumes of ethanol. The genomic DNA was then dissolved in 0.5 ml of water and stored at 48C.
2.4. Southern analysis The 0.59 kb rPTHrP cDNA probe for Southern blot analysis was labelled with [a- 32 P]dCTP using the random priming method [15]. Genomic DNA samples isolated from individual clones of rPTHrP stably-transfected MEL cells were digested with BamHI and HindIII restriction enzymes. The digested DNA samples were electrophoresed on a 0.8% agarose gel and transferred onto a Hybond N 1 filter in the presence of 0.5 N NaOH, 1.5 M NaCl. The filter was then neutralised in Tris buffer (1.5 M NaCl, 0.5 M Tris–HCl, pH 7.2) and baked at 808C for 2 h. The hybridisation was performed in 53 SSC, 53 Denhardt’s, 1% SDS and 20 mg / ml denatured herring sperm DNA at 658C overnight. The filter was washed four times in 0.13 SSC, 0.1% SDS at 608C, and exposed to an imaging plate. Bands were quantitated using the FUJIX Bio-image Analyser BAS2000 (Fuji, Photo Film Co. Ltd.).
2.5. PCR analysis 2.5.1. PCR on DNA The standard PCR reaction mixture contained 0.5 mg of genomic DNA, 0.8 mM of each primer, 100 mM of each dNTP (dATP, dGTP, dTTP, dCTP), 0.3 ml of [a- 32 P]dATP, 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 and 1.5 units of Taq DNA polymerase. All competitive PCR reactions were performed in a total volume of 50 ml and cycled in a DNA thermal cycler 4800 (Perkin-Elmer Cetus) with an overlay of mineral oil. For each set of PCR reactions, master solutions were prepared so that all reaction tubes contained the same reaction mix. The thermal cycle conditions were: 948C for 3 min (initialising step), 948C for 1 min, 608C for 1 min, 728C for 1 min (repeated for 25 cycles) and 728C for 10 min (final extension). 2.5.2. PCR on cells MEL cells were spun down and washed with TE (10 mM Tris–HCl, pH 8.3, 1 mM EDTA). Cells were resuspended in 10 ml of TE and lysed by heating to 958C for 5 min. Proteinase K was added to a concentration of 200 mg / ml. Samples were incubated at 568C for 1 h, then at 958C for 10 min to inactivate proteinase K. The entire reaction mixture from the previous step was amplified using the conditions described above, except that the MgCl 2 concentration was reduced to 1 mM. After the PCR amplification, a 15-ml aliquot of each reaction was digested with 5 units of PvuII restriction enzyme in a 30-ml volume at 378C for 2 h. Ten ml of each digested sample were electrophoresed on a 6% polyacrylamide gel in 23 TBE buffer. The gel was then dried under vacuum, and exposed to an imaging plate for 1–2 h. The imaging plate was scanned, and the data were analysed using the BAS2000.
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3. Results and discussion
3.1. Strategy of the determination of transgene copy number The procedure described for the determination of foreign gene copy number is illustrated in Fig. 1. PCR primers were designed to hybridise to regions of high sequence conservation between the rat PTHrP and mouse PTHrP genes [11,13]. The 59 and 39 primers are located at amino acids 45–52 and 130–137 of the mouse PTHrP gene exon 4 (amino acids 45–52 and 132–139 in the rat PTHrP gene) respectively. The 59 primer sequence is 100% identical for both PTHrP genes. Due to the presence of two nucleotide
Fig. 1. Procedure for determining foreign gene copy number in stably-transfected cells. The process is illustrated using murine erythroleukemia cells transformed with pLCR / EC3 / rPTHrP expression cassette. Individual steps are described in the text. * Represents a correction factor resulting from differences in dA contents of the two PCR products.
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differences in the positions of the 39 primer regions of two genes, degenerate primers comprising a mixture of two nucleotide sequences were used in order to achieve equal amplification. Genomic DNA isolated from stably-transformed MEL cells was subjected to PCR amplification in the presence of [a- 32 P]dATP. The addition of trace amounts of radioactive nucleotide allowed for the direct quantitation of PCR products after gel separation, without affecting the reaction kinetics. The amplification led to the production of a 277-bp product from the endogenous mouse PTHrP gene and a 283-bp product from the rat PTHrP transgene. The mPTHrP product contains a cleavage site for the restriction enzyme PvuII, which is absent in the rPTHrP product. Cleavage of this mouse product with PvuII yields a major fragment of 211 bp and a smaller fragment of 66 bp. Intact and digested PCR products were then separated by PAGE and the individual bands were quantitated. The recombinant gene copy number can be obtained by calculating the ratio of 283-bp and 211-bp bands (see Fig. 1). The 66-bp digested fragment of the mPTHrP product was not measured since the results would have been affected by co-migration of this fragment with other low molecular weight radioactive products, such as primer dimers. This rapid method for quantitating the copy number of a recombinant gene in stably-transfected mammalian cells can be easily applied to any transgene of known sequence. The technique utilises the single-copy endogenous gene as an internal reference standard. As the standard gene is endogenous to the genome, errors arising from the addition of external standard and quantitation of standard or sample can be avoided. We have applied the method to the determination of copy number of rat PTHrP transgene in stably-transfected mouse erythroleukemia cells.
3.2. Heteroduplex formation affected the PCR assay The initial PCR amplification was carried out with 0.3 mM of each primer and 35–40 cycles. Under the conditions used, the intact PCR products (283 bp and 277 bp) showed similar mobility on the gel electrophoresis (Fig. 2A, lane 2, band b). However, an additional band showing a slower mobility than the expected band was observed (Fig. 2A, lane 2, band a). This band remained unchanged after PvuII digestion (Fig. 2A, lane 1, band a), indicating that it has no PvuII site. To investigate whether this slow moving band was a non-specific product or a heteroduplex DNA generated by cross-hybridisation of the mPTHrP and rPTHrP products, 5 ml of PCR products were denatured by heating at 1008C for 3 min before loading on the same gel. With this treatment, both the target band and slow moving band were found to have shifted to the same position corresponding to that of a single-stranded DNA (Fig. 2A, lane 3, band d). The result strongly suggests that the slow moving band is a heteroduplex DNA. Fig. 2B shows that the heteroduplex product increased from 5% to 40% of total PCR products when PCR cycles increased from 25 to 40. Optimisation of PCR amplification conditions was carried out to reduce the formation of heteroduplex DNA. The optimum amplification conditions were achieved by increasing the concentration of PCR primers to 0.8 mM and decreasing the PCR cycle number to 25. The formation of heteroduplex DNA during the competitive PCR amplification is a factor which is likely to introduce errors into the assay. Heteroduplex DNA is resistant
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Fig. 2. Heteroduplex formation during the competitive PCR amplification of genomic DNA isolated from stably-transfected MEL cells. (A) Identification of heteroduplex product after 40 cycles of amplification. Lane 1, PCR products digested by PvuII; lane 2, PCR products; lane 3, PCR products preheated to 1008C for 3 min before being loaded on the gel. (a) Heteroduplex band; (b) 277 bp / 283 bp band resulting from amplification of the mouse and rat PTHrP genes; (c) 211 bp band from the mouse PTHrP product after the PvuII digestion; (d) single-stranded DNA band. (B) The effect of number of PCR cycles on heteroduplex formation: lane 1, 25 cycles; lane 2, 28 cycles; lane 3, 31 cycles; lane 4, 34 cycles; lane 5, 37 cycles; lane 6, 40 cycles; lane 7, negative control (no DNA template).
to restriction enzyme digestion, and therefore it is not easily distinguished from the target and competitor. The effect of heterologous duplex formation on electrophoretic mobility and the generation of heteroduplex DNA during PCR amplification have been reported previously [16,17]. According to Ruano and Kidd [17], in later PCR cycles, during which the high concentrations of PCR products are present, cross-hybridisation between products of the competitor and target gene can efficiently compete with the hybridisation of PCR primers to templates, resulting in the formation of DNA heterodimer. As the heteroduplex formed in the last PCR cycle is not denatured, it can form a significant proportion of the final products. Jenson and co-worker [18] reported a similar finding to that described here, indicating that the formation of heteroduplex DNA could be decreased by increasing PCR primer concentrations and / or by decreasing PCR cycles. Under these conditions where a high primer / template ratio is present, the formation of primer / template duplex is kinetically favoured over the rehybridisation of previously synthesised products, so that the formation of heteroduplex can be reduced.
3.3. PCR and Southern blot analyses on genomic DNA Fig. 3 illustrates the result of a PCR amplification on genomic DNA isolated from stably-transfected MEL cells and control MEL cells. In products amplified from DNA of
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Fig. 3. Polyacrylamide gel electrophoresis of co-amplified recombinant rat PTHrP gene product (283 bp) and endogenous mouse PTHrP gene product (211 bp) after 25 PCR cycles and PvuII digestion. M, markers; PCR products from MEL cells transfected with pLCR / EC3 / rPTHrP single cell clones (lanes 1–7); PCR products from MEL cells transfected with pLCR / EC3 / rPTHrP pooled populations (lanes 8–10); PCR products from control MEL cells (lane 11); negative control (no DNA template, lane 12). The rPTHrP transgene copy number: lane 1, 2; lane 2, 9; lane 3, 2; lane 4, 5; lane 5, 1; lane 6, 2; lane 7, 2; lane 8, 5; lane 9, 3; lane 10, 6; lane 11, 0.
transformed MEL cells, two bands were observed after PvuII digestion, representing the 283 bp rPTHrP product and the 211 bp mPTHrP product (lanes 1–10). In contrast, only the 211 bp mPTHrP product was observed with the DNA isolated from control MEL cells (lane 11). The latter sample also serves as a control of the PvuII digestion in our PCR assay. To test whether the PCR amplification result reflected the recombinant gene copy number present in MEL cell lines, we performed both the PCR assay and Southern blot analysis on the same DNA samples. For Southern blot analysis, the DNA samples were digested with restriction enzymes HindIII and BamHI. A Southern blot of the DNA gel was hybridised with a rat PTHrP cDNA probe. In the control sample, the probe only hybridised to the 5.1 kb fragment of mPTHrP [13], while in samples isolated from stably-transfected cells, the probe hybridised to the 0.59 kb rPTHrP fragment as well as to the 5.1 kb mPTHrP fragment. The intensity of each band was quantitated, and copy numbers of the transgenes were calculated. Table 1 shows a comparison of results of the transgene copy number determined by the competitive PCR and Southern blot analyses. Results from two different methods are consistent, indicating the reliability of PCR method for the determination of copy number of the transgene.
3.4. PCR analysis on MEL cells In several previous reports, PCR amplification has been successful using whole cells as the starting material rather than extracted DNA [3,6–8]. Hence we investigated the possibility of performing our PCR assay directly on lysed MEL cells. A range of dilutions of rPTHrP stably-transfected MEL cells was tested (from 5310 2 to 5310 5 cells per reaction). Cells were lysed by heating to 958C for 5 min, followed by a proteinase K digestion to remove contaminating proteins. Fig. 4 shows PCR products
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Table 1 Comparison of the Southern blot and PCR analyses for the determination of the rPTHrP transgene copies in MEL cells a Genomic DNA samples
Southern blot analysis
PCR assay
1 2 3 4 5 6 7 8
4.4 20.0 4.0 0.8 3.2 3.4 10.8 8.0
4.6 18.8 4.0 1.4 4.4 5.6 10.8 7.0
a DNA samples 1–6 were prepared from MEL cells transfected with pLCR / EC3 / rPTHrP single cell clones. DNA samples 7–8 were obtained from MEL cells transfected with pLCR / EC3 / rPTHrP pooled populations.
Fig. 4. Competitive PCR performed directly on whole MEL cell lysates without DNA extraction. The cells contain an average of four copies of the rPTHrP transgene. (A) Amplified PCR products. (B) PCR products digested by the PvuII restriction enzyme. Numbers of MEL cells used as templates for the reaction were as follows: lane 1, 5310 2 ; lane 2, 1310 3 ; lane 3, 5310 3 ; lane 4, 1310 4 ; lane 5, 5310 4 ; lane 6, 1310 5 ; lane 7, 5310 5 ; lane 8, negative control (no cells).
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obtained from amplification of the rat and mouse PTHrP genes from whole MEL cell lysates. Results indicated that 10 4 cells provided sufficient template to yield a positive signal after 25 amplification cycles and could be reliably used for transgene copy number determination. The sensitivity could be further increased 10-fold (i.e. 10 3 cells) if nested PCR was performed using a second pair of PCR primers (data not shown). The ability to conduct competitive PCR analysis on whole MEL cells is one of the major factors contributing to the reduction in processing time. It is important to note, however, that this method is found to be successful only if contaminating proteins are removed prior to PCR amplification by proteinase K digestion. The presence of large amounts of contaminating proteins not only reduces the efficiency of PCR amplification, but also inhibits electrophoretic mobility of the PCR products. Thus the proteinase K digestion is essential and should be conducted routinely prior to PCR amplification. The major advantage of this technique of competitive PCR over Southern blotting for determining transgene copy number is the speed with which large numbers of samples can be processed. MEL cells, like many other mammalian cell types, have a very slow growth rate, with cell divisions occurring only every 10–12 h. Screening of recombinant cell line clones by Southern blot requires at least 2 weeks for expansion of the cell line from a single cell to 10 7 cells. Subsequent DNA extraction and Southern blot analysis requires another 3–4 days. In contrast, using this competitive PCR method, PCR analysis of a cell line expanded from a single cell can take place after only 1 week. The analysis itself (including PCR amplification, restriction enzyme digestion, gel electrophoresis and band quantitation) can be completed within a day. If necessary, the process could be further streamlined by, for example, increasing the concentration of the radioactive nucleotide or utilising a more rapid thermal cycler. Thus, by screening recombinant cell lines with competitive PCR, time consuming and / or costly procedures, such as tissue culture, DNA extraction, Southern blot transfer, probe preparation and blot hybridisation, can be avoided.
4. Simplified description of the method and its (future) applications We describe here a rapid method for quantitating the copy number of a recombinant gene in stably-transfected mammalian cells that can be easily applied to any transgene of known sequence. The novel aspect of the technique is the use of the single-copy endogenous gene as an internal reference standard. The copy number is then determined by the ratio of the transgene to the endogenous gene. The transgene is differentiated from the endogenous gene by restriction enzyme cleavage of the endogenous gene. However, it should be noted that the method is only applicable when two highly homologous regions are found within the transgene and endogenous gene. In addition, we have demonstrated that the technique can be used directly on MEL cells without DNA extraction, and that a PCR of 25 cycles provides adequate signal sensitivity when 10 4 MEL cells are used. These conditions are also optimised to reduce heteroduplex formation. Since the use of mammalian expression systems to produce recombinant proteins or as a means of studying gene function is becoming more common, the ability to determine
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transgene copy number easily and rapidly will prove extremely valuable. In addition to its use in transfected cells for protein expression, this method may be applicable to measuring copy number in transgenic animals and plants.
Acknowledgements We thank Drs P. Roche and J. Gunnersen for their helpful discussions. We also thank Dr E.H. Kachab and S. Khoury for the synthesis of oligonucleotides. This work was supported by an institute grant to the Howard Florey Institute from the National Health and Medical Research Council of Australia.
References [1] Gilliland G, Perrin S, Blanchard K, Bunn HF. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci USA 1990;87:2725–9. [2] Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA 1989;86:9717–21. [3] Harlow SP, Stewart CC. Quantitation of c-myc gene amplification by a competitive PCR assay system. PCR Methods Appl 1993;3:163–8. [4] Uberla K, Platzer C, Diamantstein T, Blankenstein T. Generation of competitor DNA fragments for quantitative PCR. PCR Methods Appl 1991;1:136–9. [5] McCullock RK, Choong CS, Hurley DM. An evaluation of competitor type and size for use in the determination of mRNA by competitive PCR. PCR Methods Appl 1995;4:219–26. [6] Kawasaki ES. Sample preparation from blood, cells, and other fluids. In: Innis MA, Gelfan DH, Sninsky JJ, White TJ, editors, PCR protocols: a guide to methods and applications, San Diego, CA: Academic Press, 1990, pp. 146–52. [7] Li H, Gyllensten UB, Cui X, Saiki RK, Erilich HA, Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 1988;335:414–7. [8] Molesh DA, Hall JM. Quantitative analysis of CD34 1 stem cells using RT-PCR on whole cells. PCR Methods Appl 1994;3:278–84. [9] Needham M, Gooding C, Hudson K, Antoniou M, Grosveld F, Hollis M. LCR / MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells. Nucl Acids Res 1992;20:997–1003. [10] Talbot D, Collis P, Antoniou M, Vidal M, Grosveld F, Greaves DR. A dominant control region from the human beta-globin locus conferring integration site-independent gene expression. Nature 1989;338:352– 5. [11] Yasuda T, Banville D, Rabbani SA, Hendy G, Goltzman D. Rat parathyroid hormone-like peptide: comparison with the human homologue and expression in malignant and normal tissue. Mol Endocrinol 1989;3:518–25. [12] Fourney RM, Aubin R, Dietrich KD, Paterson MC. Determination of foreign gene copy number in stably transfected cell lines by Southern transfer analysis. In: Murray EJ, editor, Methods in molecular biology: gene transfer and expression protocols, Vol. 7, Clifton, NJ: The Human Press, 1991, pp. 381–95. [13] Mangin M, Ikeda K, Broadus AE. Structure of the mouse gene encoding parathyroid hormone-related peptide. Gene 1991;95:195–202. [14] Antoniou M. Induction of erythroid-specific expression in murine erythroleukemia (MEL) cell lines. In: Murray EJ, editor, Methods in molecular biology: gene transfer and expression protocols, Vol. 7, Clifton, NJ: The Human Press, 1991, pp. 421–34. [15] Sambrook J, Fritsch EF, Maniatis T. 2nd ed, Molecular cloning: a laboratory manual, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1989.
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P. Fu et al. / J. Biochem. Biophys. Methods 40 (1999) 101 – 112
[16] Hsiech CH, Griffith JD. Deletions of bases in one strand of duplex DNA, in contrast to single-base mismatches, produce highly kinked molecules: possible relevance to the folding of single-stranded nucleic acids. Proc Natl Acad Sci USA 1989;86:4833–7. [17] Ruano G, Kidd KK. Modeling of heteroduplex formation during PCR from mixtures of DNA templates. PCR Methods Appl 1992;2:112–6. [18] Jenson MA, Strano N. Effect of PCR conditions on the formation of heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacer regions. PCR Methods Appl 1993;3:186–94.