A novel method for the direct quantification of gene transfer into cells using PCR in situ

Catzavelos, Charles; Ruedy, Christine; Ak, Shalek; Dubé, Ian D.

doi:10.1038/sj.gt.3300663

Cited by 3 publications

(2 citation statements)

References 24 publications

(33 reference statements)

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“…9,10 However, quantification of the presence and/or expression necessitates labor and time-consuming methods such as competitive PCR, either alone 11 or in combination with ELISA detection 12 or with fluorescence in situ hybridization. 13 Recently, a new technology based on the 5Ј-3Ј exonuclease activity of the Taq DNA polymerase 14 has been used to monitor gene transfer efficiency into hematopoietic cells. 15 This method utilizes the 5Ј nuclease activity of the Taq polymerase to cleave a fluorogenic probe.…”

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

et al. 2000

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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 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 CD24 2 or the green fluorescent protein [3][4][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 gene 7,8 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 alone 11 or in combination with ELISA detection 12 or with fluorescence in situ hybridization. 13 Recently, a new technology based on the 5Ј-3Ј exonuclease activity of the Taq DNA polymerase 14 has been used to monitor gene transfer efficiency into hematopoietic cells.15 This method utilizes the 5Ј nuclease activity of the Taq polymerase to cleave a fluorogenic probe. The resulting fluorescence is proportional to the 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. amount of amplified DNA and can be measured directly after PCR, thus avoiding labor-intensive post-PCR steps. This te...

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confidence: 99%

“…13 Recently, a new technology based on the 5Ј-3Ј exonuclease activity of the Taq DNA polymerase 14 has been used to monitor gene transfer efficiency into hematopoietic cells.…”

mentioning

confidence: 99%

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

et al. 2000

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Application of In Situ PCR to Studies of the Mammary Gland

Hovey

Vonderhaar

2000

Methods in Mammary Gland Biology and Breast Cancer Research

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Improvement of In Situ PCR by Optimization of PCR Cycle Number and Proteinase K Concentration: Localization of X Chromosome-Linked Phosphoglycerate Kinase-1 Gene in Mouse Reproductive Organs

Hishikawa

Yamamoto‐Fukuda

et al. 2009

Acta Histochem. Cytochem

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In situ polymerase chain reaction (in situ PCR), which can detect a few copies of genes within a cell by amplifying the target gene, was developed to better understand the biological functions of tissues. In this study, we optimized the protocol conditions for the detection of X chromosome-linked phosphoglycerate kinase-1 (pgk-1) gene in paraffin-embedded sections of mouse reproductive organs. The effects of various concentrations of proteinase K (PK) and PCR cycle numbers were examined. To label the amplified DNA, we used digoxigenindUTP (Dig), Cy-3-dUTP (Cy-3), or FluorX-dCTP (FluorX). The optimal concentration of PK was 50 µg/ml for the ovary and 10 µg/ml for the testis. Ten PCR cycles were optimal for Dig and 25 cycles were optimal for FluorX and Cy-3 in the ovary and testis. The signal-to-noise ratio of FluorX and Cy-3 for ovarian tissue was better than that of Dig. Using the above conditions, we detected 1-4 and 1-2 spots of pgk-1 in the nuclei of granulosa and germ cells, respectively. Our results indicate that in situ PCR is useful for detecting a specific gene in paraffin-embedded sections under optimized conditions of both PCR cycle number and PK concentration.

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A novel method for the direct quantification of gene transfer into cells using PCR in situ

Cited by 3 publications

References 24 publications

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

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

Application of In Situ PCR to Studies of the Mammary Gland

Improvement of In Situ PCR by Optimization of PCR Cycle Number and Proteinase K Concentration: Localization of X Chromosome-Linked Phosphoglycerate Kinase-1 Gene in Mouse Reproductive Organs

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