Accurate estimation of transduction efficiency necessitates a multiplex real-time PCR

Gene Therapy (2000) 7, 458–463  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt

VIRAL TRANSFER TECHNOLOGY

TECHNIQUES

Accurate estimation of transduction efficiency necessitates a multiplex real-time PCR D Klein1, B Bugl2, WH Gu¨nzburg1 and B Salmons2

Institute of Virology, University of Veterinary Sciences, Vienna, Austria; and 2Bavarian Nordic Research Institute GmbH, Martinsried, Germany

1

Transduction efficiency can be easily monitored during preclinical trials by inclusion of marker genes. However, the use of such marker genes should be avoided in the final clinical gene therapy application since their products are often immunogenic, making it difficult to monitor transduction, especially if the vector is applied in vivo. In these cases PCR-based methods like the real-time PCR might provide a powerful tool to estimate biodistribution. To investigate the accuracy of this method, we have developed and tested a real-time PCR assay for the quantification of the enhanced green fluorescent protein (EGFP) gene and compared the results with transduction efficiencies estimated by FACS

analysis. Although our real-time PCR assay itself was characterized by a high precision over a wide dynamic range of quantification, significant differences in the transduction efficiency compared with FACS data were initially observed. Accurate determination could only be achieved using an optimized multiplex real-time PCR assay, which allows the simultaneous calculation of cell number and EGFP copy number in the same tube. In view of future needs for methods allowing precise and accurate analysis of biodistribution in gene therapy trials, our data highlight the necessity critically to check both parameters in the implemented assay. Gene Therapy (2000) 7, 458–463.

Keywords: real-time PCR; retroviral vector; transduction efficiency; TaqMan; gene therapy

During the past 10 years the number of clinical gene therapy studies has increased exponentially. Unfortunately, most of these studies suffer from low transduction or expression efficiencies. Introduction of new marker genes such as truncated nerve growth factor,1 CD242 or the green fluorescent protein3–5 represented an important step in the development of improved gene transfer protocols since transduction efficiency could be easily monitored using flow cytometry.6 However, in the final in vivo gene therapy situation such marker genes have to be avoided to prevent the induction of an immune response against the marker gene7,8 and therefore improve the safety and efficacy of these vectors. In comparison, transduction of therapeutic genes cannot usually be monitored easily by flow cytometry. In cases where an indication of the presence of the vector suffices, standard PCR technologies can be used.9,10 However, quantification of the presence and/or expression necessitates labor and time-consuming methods such as competitive PCR, either alone11 or in combination with ELISA detection12 or with fluorescence in situ hybridization.13 Recently, a new technology based on the 5!–3! exonuclease activity of the Taq DNA polymerase14 has been used to monitor gene transfer efficiency into hematopoietic cells.15 This method utilizes the 5! nuclease activity of the Taq polymerase to cleave a fluorogenic probe. The resulting fluorescence is proportional to the

Correspondence: D Klein, Institute of Virology, University of Veterinary Sciences, Veterina¨rplatz 1, A-1210 Vienna, Austria Received 13 September 1999; accepted 8 November 1999

amount of amplified DNA and can be measured directly after PCR, thus avoiding labor-intensive post-PCR steps. This technology has been further developed to measure released fluorescence during PCR (real-time PCR), which allows the estimation of the input copy number during the exponential phase of the reaction and enables estimation of transduction efficiencies over a wide range of quantification.16 The same technology has been utilized to monitor expression levels of transduced genes17 and for the rapid determination of number of retroviral particles.18 Beside the demonstrated advantages of the realtime PCR technology (high sensitivity, wide dynamic range of quantification, no post-PCR steps), the precision and accuracy of the estimated transduction efficiency are critical for its utilization in biodistribution investigations. The precision of the real-time PCR method itself has been demonstrated by low intra- and inter-assay variations,16,17,19 whereas the precision and the accuracy (the correctness of the final result) of the complete assay in comparison to commonly used methods has not been investigated. In order to investigate these two important aspects of quantitative real-time PCR assays for the estimation of transduction efficiency, we have designed primers and a fluorescent probe for the detection and quantification of the EGFP gene. This approach enabled us to compare the precision and accuracy of the real-time PCR with the data obtained by FACS analysis. The primer and probe sequences were designed with Primer Express software (Perkin-Elmer, Foster City, CA, USA). The primers of the EGFP734p assay were EGFP553f (5!-ATC ATG GCC GAC AAG CAG AAG AAC-3!) and EGFP810r (5!-GTA CAG CTC GTC CAT

Transduction efficiency by multiplex real-time PCR D Klein et al

GCC GAG AGT-3!). The probe used in this system was EGFP734p (5!-FAM-CAG GAC CAT GTG ATC GCG CTT CTC GT-TAMRA-3!). The 50 "l PCR mixtures contained 10 mm Tris (pH 8.3), 50 mm KCl, 3 mm MgCl2, 200 nm dATP, dCTP, dGTP, 400 nm dUTP, 300 nm of each primer,200 nm of the fluorogenic probe and 2.5 units of Taq DNA polymerase. After the initial denaturation (2 min at 95°C), amplification was performed with 45 cycles of 15 s at 95°C and 60 s at 60°C. Amplification was performed in an ABI Prism 7700 Sequence Detection System (Perkin-Elmer). The probes are chosen to hybridize to an internal sequence of the PCR target. After excitation with an argon laser, the energy from the reporter fluorochrome (6-carboxy-fluorescein, FAM) is transferred to the quencher fluorochrome (6-carboxy-tetramethylrhodamine, TAMRA) and only the quencher emits sufficient amounts of light. The quenching effect occurs only as long as both fluorochromes are present as part of the intact probe. During the elongation of the complementary strands, the probe is displaced and cleaved by the 5!–3!-

nuclease activity of the Taq polymerase, thus eliminating the quenching by the separation of the two fluorochromes. The resulting increase in the reporter fluorescence is proportional to the amount of amplified product during the PCR and is measured every 7 s in the 96well format. In the first step of the validation procedure of the realtime PCR system the dynamic range of quantification and the sensitivity of the assay was tested. For this purpose a 10-fold dilution series of the plasmid pLXSNEGFP4 containing the EGFP gene was used (Figure 1a). The limit of detection was five copies, where two out of three measurements were positive. The linear range of quantification was seven logarithmic decades with a coefficient of correlation (r2) of 0.9965 for the triplicate measurements. Another important parameter of the system, the reaction efficiency, can be obtained from a standard curve.19 A high coefficient of correlation (r2 # 0.995) is a prerequisite to allow the calculation of the reaction efficiency (E) from the slope of this standard dilution series (a detailed description of the equation has previously

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Figure 1 Standard dilution series of the different real-time PCR systems. (a) The plasmid pLXSNEGFP4 which contains the EGFP gene under the control of the LTR, as well as a SV40 neomycin expression cassette, was used as a standard for the estimation of the sensitivity and linear range for quantification of the EGFP734p real-time PCR system. The copy number was calculated after OD measurement at 260 nm. Ten-fold dilution series were made in PCR grade water containing 30 "g calf thymus DNA per ml as carrier DNA and measured in triplicate. The resulting standard curve is illustrated by the mean and the standard deviation of each dilution step. The coeffiecient of correlation (r2), slope and reaction efficiency (E) are indicated in the box. The resulting PCR products were visulized after gel electrophoresis seperation on a 1.5% agarose gel. (b) The EGFP734p realtime PCR system was tested using a genomic DNA standard obtained from NIH/3T3 cells stably transduced with the LXSNEGFP retroviral vector and extracted using the QIAamp Kit (Qiagen). The DNA concentration was estimated by OD260 measurement and a four-fold dilution series was made in TE buffer containing 3 "g t-RNA per ml. (c) The rDNA real-time PCR system was tested with the same genomic DNA dilution series as in (b). (d) The new EGFP234v real-time PCR system was tested with the same genomic DNA dilution series as in (b) and (c).

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Table 1 Different amounts of the pLXSNEGFP plasmid were measured 11 times in one EGFP734p real-time PCR Copy number 5 × 101 5 × 102 5 × 104 5 × 107

CVCT value %

CVcopy number %

1.15 2.58 1.13 1.09

27.10 34.51 17.76 10.74

The coefficients of variation are calculated either from the resulting threshold cycle values (CVCT) or the extrapolated copy numbers (CVcopy number).

been described19). In this case the calculated efficiency was 0.9320. In order to analyze the specificity of the primers, the PCR product was visualized on an agarose gel (Figure 1a). Only the expected band at 258 bp and no additional bands can be seen on the gel. In the negative control a shorter band was detected. However, this shorter PCR product is not detected by the TaqMan probe, thus demonstrating the higher specificity of the real-time PCR due to the in reactio hybridization of the probe. To investigate the influence on reaction efficiency of the integration of the target gene into genomic DNA, a four-fold dilution series of genomic DNA containing the EGFP gene integrated into NIH/3T3 cells was analyzed in duplicate (Figure 1b). A positive signal was obtained in the dilution step containing genomic DNA from five cells. The resulting standard curve was nearly linear (r2: 0.9971) over five logarithmic decades. The high coefficient of correlation met the criteria defined above and allowed the calculation of the reaction efficiency (E: 0.8919) from the slope (−3.611). A similar reduction in reaction efficiency has been previously found using a real-time PCR system targeting a different gene.19 However, this minor reduction in reaction efficiency from 0.9320 to 0.8919 did not affect sensitive quantification of the target gene, and enabled us to use a dilution series of genomic DNA rather than the more commonly used plasmid dilution series20 as an external standard. This is

especially important since a genomic DNA standard reflects more accurately the situation in the tested samples. To investigate whether this decreased reaction efficiency is due to unspecific binding of primers, the PCR products were visualized on an agarose gel. Only the the expected band at 258 bp and no additional bands were visible on the gel (Figure 1b), suggesting that the observed reduction in reaction efficiency is due to the higher complexity of genomic DNA compared with plasmid DNA. Another important step in the validation of the realtime PCR assay was the investigation of the reproducibility of measurements. For this purpose different amounts of target DNA were measured 11 times within one run and the mean and standard deviation of these replicate measurements was calculated (Table 1a). The coefficient of variation (CV) of the threshold cycle (CT) varied between 1.09% and 2.58%. However, these logarithmic CT values have to be extrapolated into linear copy numbers, which increases the variation of the values (Table 1b) – a general feature of all PCR-based quantification methods. After demonstrating that the precision of our real-time PCR assay is similar to that previously described,16,19,20 we were interested in the accuracy of the real-time PCR data compared with data obtained from FACS analysis, a standard method for the estimation of transduction efficiency. FACS analysis was chosen since in both methods the transduction efficiency is calculated as the percentage of positive cells and the FACS analyses reflect a more objective estimation of transduction efficiency independent of laboratory-specific variations (eg postinfection plating efficiency, trypsinization procedures etc). For this purpose a LXSN-based retroviral vector21 containing the EGFP gene under the control of the retroviral LTR4 was used to transduce NIH/3T3 cells. The transduction efficiency was estimated by FACS analysis at several time-points after infection (Figure 2a and b) and remained stable for at least 60 days, a phenomenon previously reported.4 At the same time-points an aliquot of infected cells was

Figure 2 Influence of cell number estimation on transduction efficiency by real-time PCR. Twenty-four hour conditioned cell culture medium from 5 × 105 LXSNEGFP vector producing cells was passed through a 0.45 "m Millipore filter, polybrene added to a final concentration of 8 "g/ml, and 2 ml used to transduce 5 × 105 NIH/3T3 target cells in a 10 cm culture dish. After 6 h incubation, 6 ml of DMEM medium containing 10% FCS was added and the cells cultured further. The percentage of EGFP-positive cells were analyzed in triplicate measurements at different time-points after infection using a FACSCalibur (Becton Dickinson, Heidelberg, Germany). At the same time-points an aliquot of cells was used for extraction of genomic DNA (QIAamp Kit). The EGFP copy number per 5 "l genomic DNA was estimated in duplicate using the EGFP734p real-time PCR. The cell number per 5 "l genomic DNA was estimated either by OD measurement at 260 nm (a) or by a second real-time PCR assay targeting the rDNA genes (b). The calculated transduction efficiency is illustrated by the mean and the standard deviation for each time-point. Gene Therapy

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Figure 3 Influence of the multiplex setup on the reaction efficiency. Genomic DNA from NIH/3T3 cells stably transduced with the LXSNEGFP retroviral vector was extracted using the QIAamp Kit (Qiagen). The DNA concentration was estimated by OD260 measurement and a four-fold dilution series was made in TE buffer containing 3 "g t-RNA per ml. The dilution series was measured in duplicate with a rDNA monoplex real-time PCR ((a) monoplex), a EGFP234v real-time PCR ((b) monoplex) and a 300 nm EGFP234v/rDNA multiplex real-time PCR with primer ratio of 300 nm to 60 nm ((a) multiplex, (b) multiplex and (d)). The resulting standard curves are illustrated using the mean and the standard deviation for each dilution step. The corresponding coefficients of correlation (r2), the slope of the standard curves and the calculated reaction efficiencies (E) are listed in (c).

used for genomic DNA extraction (QIAamp, Qiagen, Hilden, Germany). Each sample was measured in duplicate in the real-time PCR and the EGFP copy number per reaction was determined using an external standard curve of genomic DNA. The number of cells per PCR reaction was estimated by an OD measurement at 260 nm, assuming a mean DNA content of 6 pg per cell. The resulting transduction efficiency at the different timepoints was compared with the results obtained by FACS analysis (Figure 2a). Surprisingly, the calculated transduction efficiency obtained by real-time PCR in conjunction with OD measurement displayed a high degree of inaccuracy (three to eight times higher values) and variation (high standard deviation). This finding was not expected since the precision of the real-time PCR and OD measurement were validated and a generally accepted method for isolation of genomic DNA and subsequent PCR was used. Other factors, which influence the accuracy of the real-time PCR data include large discrepancies between the integrated copy number of the standard and the sample to be measured, as well as unacceptably large variations in the signal measured from the external standard. These points have been addressed: (1) by using a genomic standard infected with a similar MOI as the samples and (2) by ensuring that the coefficients of correlation of the standard curves are #0.99 and have similar slopes and CT values. The most likely explanation for this discrepancy between the real-time data corrected by OD measurements versus the FACS data would be that the quality of the sample DNA has a different influence on

estimation of cell number by OD measurement than on the transduced gene quantification by real-time PCR. In order to minimize the influence of DNA quality on transduction efficiency calculation, a second real-time PCR assay targeting the 18S rDNA genes was developed. In this setup the quality of the sample DNA should have the same influence on the cell number as on the transferred gene estimation and therefore improve the accuracy of the complete transduction efficiency calculation. The rDNA assay consists of the primers rDNA343f (5!CCA TCG AAC GTC TGC CCT A-3!) and rDNA409r (5!TCA CCC GTG GTC ACC ATG-3!) and the probe rDNA370p (5!-FAM-CGA TGG TGG TCG CCG TGC CTA-TAMRA-3!). The system was tested with the above described four-fold dilution series of genomic DNA containing the EGFP gene integrated in NIH/3T3 cells (Figure 1c). The standard curve was linear in the relevant range of 5 to 5 × 105 cells with a high coefficient of correlation of 0.9992. This high CV allowed the calculation of a reaction efficiency of 0.9512 from the slope (−3.445). To test whether the use of two PCR assays for cell number and gene transfer calculation would improve the accuracy of the system, the same samples as in Figure 2a were analyzed in the rDNA real-time PCR system. The resulting cell numbers were used to calculate the percentage infected cells from the EGFP copy numbers (Figure 2b). The overall results obtained from the combination of the two real-time PCRs was more or less in the range of the data obtained by FACS analysis, reflecting the higher accuracy of cell number estimation by real-time PCR Gene Therapy

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compared with OD measurement. Nevertheless, two time-points showed inconsistency: early after infection and at day 40. The two-fold higher values measured by real-time PCR as compared with FACS analysis early after infection might be due to reverse transcribed but not expressed vector genomes, since unintegrated DNA represents more than 75% of the viral DNA after transduction.22 However, the five-fold higher value measured at day 40 could not be explained by this theory and might reflect the accumulation of errors (eg pipetting errors of sample and external standard in conjunction with minimal inter-assay variations and the transformation from logarithmic PCR values into linear copy numbers; see Table 1) in multiple PCRs necessary in this setup. To circumvent this problem and minimize variations, we developed a multiplex real-time PCR for simultaneous measurement of the EGFP copy number and the cell number in a one-tube assay. For this purpose a new real-time PCR system for the EGFP gene, which has a similar amplicon length and does not have a predicted dimer formation with the rDNA components, was designed. This new EGFP real-time PCR system has to be labelled with a different reporter dye (VICTM) to enable the differentiation of the two targets and consisted of the probe EGFP234v (5!-VIC-CCG ACC ACA TGA AGC AGC ACG ACT T-3!-TAMRA) and the primers EGFP214f (5!-GCA GTG CTT CAG CCG CTA C-3!) and EGFP309r (5!-AAG AAG ATG GTG CGC TCC TG-3!). To test this system the same 1:4 dilution series of genomic DNA from the NIH/3T3-based cell line as in Figure 1b and c was used. A linear range for quantification of five logarithmic decades with a high coefficient of correlation (r2) of 0.9974 and a sensitivity of at least 5 copies (Figure 1d) was obtained. The higher reaction efficiency of the new compared with the old EGFP real-time PCR system (1.0235 compared with 0.8919) is probably due to the shorter amplicon length in this system (96 bp compared with 258 bp). Nevertheless, the amplicon length is not the only factor which influences the reaction efficiency, since the new EGFP real-time PCR system has a longer amplicon length than the rDNA real-time PCR system (96 bp compared with 67 bp), but a better reaction efficiency. The observed reaction efficiencies and amplicon lengths from the new EGFP and the rDNA real-time PCR systems suggest good conditions for the setup of a multiplex system, in which both systems compete for common reagents.

To limit the reaction of the more abundant rDNA target in the multiplex real-time PCR, the primer concentration for this target has to be reduced. The lowest rDNA primer concentrations necessary for accurate rDNA quantification were determined using a matrix of reactions, each with different concentrations of the rDNA forward and reverse primers in a monoplex real-time PCR. The CT values are similar in the range from 100 nm down to 60 nm for both primers and start to increase below a primer concentration of 60 nm (data not shown), while the $Rn value is much lower at 60 nm compared with 300 nm (data not shown). This data suggests that at 60 nm the primer concentration limits the rDNA PCR without affecting the reaction efficiency in the exponential phase of the reaction. In order to investigate the influence of the primer reduction of the rDNA system in the multiplex setup, a 1:4 serial dilution of genomic DNA from EGFP bearing NIH/3T3 cells in TE buffer using t-RNA as the carrier was measured with a dilution series containing decreasing amounts of both EGFP copy number and cell number. This setup allows the simultaneous estimation of reaction efficiency of both EGFP and rDNA in the mono- and multiplex setup. The multiplex setup resulted in a steeper slope of the standard curve (Figure 3a and b) and thus a slightly decreased reaction efficiency (rDNA 0.8561 to 0.7685; EGFP 1.0202 to 0.9275; Figure 3c), for both systems. Nevertheless, the precise quantification of both target genes in the multiplex PCR was not impaired, since both standard curves display a linear curve over the relevant area of four logarithmic decades (Figure 3d). After the demonstration of the sensitivity and the precision of the multiplex real-time PCR for both targets we investigated whether the accuracy of the transduction efficiency determination was improved. NIH/3T3 cells were transduced with the LXSNEGFP vector in two independent experiments. The transduction efficiency was determined 48 h after transduction by FACS analysis and compared with the the data obtained by different realtime PCR assays (Figure 4). In both experiments the calculated transduction efficiency determined by the EGFP/rDNA multiplex real-time PCR approach was consistent with the FACS data (Figure 4: black bars; shaded bars), while the results obtained by two independent monoplex real-time PCRs (one for EGFP copy number and the other for cell number determination) varied dramatically (Figure 4: first open bars). The monoplex

Figure 4 Accuracy of transduction efficiency estimated by multiplex and monoplex real-time PCR. 5 × 105 NIH/3T3 cells were transduced with the LXSNEGFP vector (two independent experiments) and FACS titer was estimated 48 h after transduction (quadruplicate measurements; black bars). At the same time-point an aliquot of cells was used for extraction of genomic DNA. The real-time PCR-derived transduction efficiency was either estimated by a EGFP234v/rDNA multiplex real-time PCR (shaded bars) or by two monoplex real-time PCR assays (either EGFP234v and rDNA (open bars) or Neo423p and rDNA (striped bars)). The calculated transduction efficiency is illustrated using the mean and the standard deviation. Gene Therapy

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approach was then repeated and displayed a similar variation (Figure 4: second open bar). The observed results from the monoplex approach are in accordance with the results from Figure 2b and confirmed the suggestion that an accumulation of errors in the two independent monoplex real-time PCR assays are responsible for the observed variation and can be overcome by a multiplex approach. Nevertheless, to exclude a target specific phenomenon, a real-time PCR targeting the second marker gene of the LXSNEGFP vector, the Neomycin phosphotransferase (Neo) gene, was used additionally. The Neo real-time PCR system consisted of the primers Neo382f (5!-CCG GCT ACC TGC CCA TTC-3!) and Neo456r (5!-AAG ACC GGC TTC CAT CCG-3!) and the probe Neo423p (5!-FAM-AAC ATC GCA TCG AGC GAG CAC GT-TAMRA-3!). The estimated transduction efficiency displayed a similar inconsistency compared with the FACS and multiplex real-time PCR data (Figure 4: striped bars), suggesting that the monoplex approach enables only an estimation but not an accurate determination of transduction efficiencies or retroviral titers. In addition, these results highlight the necessity to check critically the accuracy of the assay irrespective of assay precision. In conclusion, several real-time PCR approaches have been developed to determine transduction efficiency. The results obtained were compared with FACS data and demonstrated that an optimized real-time PCR with a high demonstrated precision does not guarantee accurate estimation of transduction efficiency. The accuracy depends on the method used for cell number estimation and can be further optimized by using a multiplex realtime PCR approach, which allows simultaneous quantification of the transduced gene and of the cell number. In view of urgent needs for new methods to estimate transduction efficiency in cases where no marker genes have been used, our data highlight the necessity to test critically the accuracy and precison of the assay used.

References 1 Mavilio F et al. Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer. Blood 1994; 83: 1988– 1997. 2 Pawliuk R, Eaves CJ, Humphries K. Sustained high-level reconstitution of the hematopoietic system by preselected hematopoietic cells expressing a transduced cell-surface antigen. Hum Gene Ther 1997; 8: 1595–1604. 3 Levy JP, Muldoon RR, Zolotukhin S, Link CJ. Retroviral transfer and expression of a humanized, red-shifted green fluorescent protein gene into human tumor. Nat Biotechnol 1996; 14: 610–614. 4 Klein D et al. Rapid identification of viable retrovirus-trans-

duced cells using the green fluorescent protein as a marker. Gene Therapy 1997; 4: 1256–1260. 5 Levenson VV, Transue ED, Roninson IB. Internal ribosomal entry site-containing retroviral vectors with green fluorescent protein and drug resistance markers. Hum Gene Ther 1998; 9: 1233–1236. 6 Brenner M. Gene marking. Hum Gene Ther 1996; 7: 1927–1936. 7 Riddell SR et al. T-cell mediated rejection of gene-modified HIVspecific cytotoxic T lymphocytes in HIV-infected patients. Nature Med 1996; 2: 216–223. 8 Stipecke R et al. Immune response to green fluorescent protein: implications for gene therapy. Gene Therapy 1999; 6: 1305–1312. 9 Long Z et al. Biosafety monitoring of patients receiving intracerebral injections of murine retroviral vector producer cells. Hum Gene Ther 1998; 9: 1165–1172. 10 Long Z et al. Molecular evaluation of biopsy and autopsy specimens from patients receiving in vivo retroviral gene therapy. Hum Gene Ther 1999; 10: 733–740. 11 Tafuro S, Zentilin L, Falaschi A, Giacca M. Rapid retrovirus titration using competitive polymerase chain reaction. Gene Therapy 1996; 3: 679–684. 12 Lahijani R et al. Quantitation of host cell DNA contaminate in pharmaceutical-grade plasmid DNA using competitive polymerase chain reaction and enzyme-linked immunosorbent assay. Hum Gene Ther 1998; 9: 1173–1180. 13 Catzavelos C, Ruedy C, Stewart AK, Dube I. A novel method for the direct quantification of gene transfer into cells using PCR in situ. Gene Therapy 1998; 5: 755–760. 14 Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5!–3! exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA 1991; 88: 7276–7280. 15 Gerard CJ et al. A rapid and quantitative assay to estimate gene transfer into retrovirally transduced hematopoietic stem/progenitor cells using a 96-well format PCR and fluorescent detection system universal for MMLV-based proviruses. Hum Gene Ther 1996; 7: 343–354. 16 Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996; 6: 986–994. 17 Gibson UE, Heid CA, Williams PM. A novel method for real time quantitative RT-PCR. Genome Res 1996; 6: 995–1001. 18 Sanburn N, Cornetta K. Rapid titer determination using quantitative real-time PCR. Gene Therapy 1999; 6: 1340–1345. 19 Klein D et al. Proviral load determination of different feline immunodeficiency virus isolates using real-time polymerase chain reaction: influence of mismatches on quantification. Electrophoresis 1999; 20: 291–299. 20 Paradis K et al. Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. Science 1999; 285: 1236–1241. 21 Miller AD, Rosman GJ. Improved retroviral vectors for gene transfer and expression. Biotechniques 1989; 7: 980–990. 22 Varmus HE, Heasley S, Linn J, Wheeler K. Use of alkaline sucrose gradients in a zonal rotor to detect integrated and unintegrated avian sarcoma virus-specific DNA in cells. J Virol 1976; 18: 574–585.

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