Transgene Detection by Digital Droplet PCR

Moser, Dirk; Braga, Luca; Raso, Andrea; Zacchigna, Serena; Giacca, Mauro; Simon, Perikles

doi:10.1371/journal.pone.0111781

Cited by 34 publications

(36 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A novel technology, ddPCR, was recently developed to enable the absolute quantification of nucleic acid target sequences. PCR with a TaqMan probe is performed within each droplet containing fragmented DNA, and the presence of the target gene is determined by counting the number of fluorescent signal-positive droplets 23, 24, 25. Based on this strategy, we established a new method for the absolute quantification of cells expressing the target gene.…”

Section: Discussionmentioning

confidence: 99%

Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy

Igarashi

Uchiyama

Minegishi

et al. 2017

Molecular Therapy - Methods & Clinical Development

View full text Add to dashboard Cite

Clinical improvement in stem cell gene therapy (SCGT) for primary immunodeficiencies depends on the engraftment levels of genetically corrected cells, and tracing the transgene in each hematopoietic lineage is therefore extremely important in evaluating the efficacy of SCGT. We established a single cell-based droplet digital PCR (sc-ddPCR) method consisting of the encapsulation of a single cell into each droplet, followed by emulsion PCR with primers and probes specific for the transgene. A fluorescent signal in a droplet indicates the presence of a single cell carrying the target gene in its genome, and this system can clearly determine the ratio of transgene-positive cells in the entire population at the genomic level. Using sc-ddPCR, we analyzed the engraftment of vector-transduced cells in two patients with severe combined immunodeficiency (SCID) who were treated with SCGT. Sufficient engraftment of the transduced cells was limited to the T cell lineage in peripheral blood (PB), and a small percentage of CD34+ cells exhibited vector integration in bone marrow, indicating that the transgene-positive cells in PB might have differentiated from a small population of stem cells or lineage-restricted precursor cells. sc-ddPCR is a simplified and powerful tool for the detailed assessment of transgene-positive cell distribution in patients treated with SCGT.

show abstract

Section: Discussionmentioning

confidence: 99%

Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy

Igarashi

Uchiyama

Minegishi

et al. 2017

Molecular Therapy - Methods & Clinical Development

View full text Add to dashboard Cite

show abstract

“…By developing real‐time PCR tests to target exon‐exon junctions in cDNA, it has been shown that it is possible to detect very low amounts of a transgene in blood samples . A similar approach was employed by Moser et al in the development of a droplet digital PCR‐based method to detect an IGF1 transgene . At present, PCR methods hold the most promise for gene doping detection, although this approach requires design, meticulous optimisation and validation of a method specific for each target gene.…”

Section: Detectionmentioning

confidence: 99%

Equine performance genes and the future of doping in horseracing

Wilkin

Baoutina

Hamilton

2017

Drug Testing and Analysis

View full text Add to dashboard Cite

A horse's success on the racetrack is determined by genetics, training and nutrition, and their translation into physical traits such as speed, endurance and muscle strength. Advances in genetic technologies are slowly explaining the roles of specific genes in equine performance, and offering new insights into the development of novel therapies for diseases and musculoskeletal injuries that cause early retirement of many racehorses. Gene therapy approaches may also soon provide new means to artificially enhance the physical performance of racehorses. Gene doping, the misuse of gene therapies for performance enhancement, is predicted to be the next phase of doping faced by horseracing. The risk of gene doping to human sports has been recognised for almost 15 years, and the introduction of the first gene doping detection tests for doping control in human athletes is imminent. Gene doping is also a threat to horseracing, but there are currently no methods to detect it. Efficient and accurate detection methods need to be developed to deter those looking to use gene doping in horses and to maintain the integrity of the sport. Methods developed for human athletes could offer an avenue for detection in racehorses. Development of an equine equivalent test will first require identification of equine genes that will likely be targeted by gene doping attempts. This review focuses on genes that have been linked to athletic performance in horses and, therefore, could be targeted for genetic manipulation. The risks associated with gene doping and approaches to detect gene doping are also discussed.

show abstract

“…[31] The method allowed the detection of IGF-I transgenic elements for a 33-day period in DNA extracted from whole blood of 6 mice injected intramuscularly with AAV9-IGF-I elements. A duplex ddPCR protocol for IGF-I and EPO transgene elements was further developed to simultaneously detect two candidate genes for gene doping in a single assay.…”

Section: Detection Of Gene Dopingmentioning

confidence: 99%

“…Moreover, ddPCR can handle large amounts of background DNA in the reaction and is more tolerant to inhibitors and variation in amplification efficiencies. [31] These researches highlight the great effort that has been invested on the development of direct detection method of gene doping. It also demonstrates the will of anti-doping authorities to operate in a proactive way.…”

Section: Detection Of Gene Dopingmentioning

confidence: 99%

Loop‐mediated isothermal amplification (LAMP) as an alternative to PCR: A rapid on‐site detection of gene doping

Salamin

Kuuranne

Saugy

et al. 2017

Drug Testing and Analysis

View full text Add to dashboard Cite

Innovation in medical research has been diverted at multiple occasions to enhance human performance. The predicted great progress in gene therapy has raised some concerns regarding its misuse in the world of sports (gene doping) for several years now. Even though there is no evidence that gene doping has ever been used in sports, the continuous improvement of gene therapy techniques increases the likelihood of abuse. Therefore, since 2004, efforts have been invested by the anti-doping community and WADA for the development of detection methods. Several nested PCR and qPCR-based strategies exploiting the absence of introns in the transgenic DNA have been proposed for the long-term detection of transgene in blood. Despite their great sensitivity, those protocols are hampered by limitations of the techniques that can be cumbersome and costly. The purpose of this perspective is to describe a new approach based on loop-mediated isothermal amplification (LAMP) for the detection of gene doping. This protocol enables a rapid and simple method to amplify nucleic acids with a high sensitivity and specificity and with a simple visual detection of the results. LAMP is already being used in clinical application for the detection of viruses or mutations. Therefore, this technique has the potential to be further developed for the detection of foreign genetic material in elite athletes.

show abstract

Transgene Detection by Digital Droplet PCR

Cited by 34 publications

References 29 publications

Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy

Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy

Equine performance genes and the future of doping in horseracing

Loop‐mediated isothermal amplification (LAMP) as an alternative to PCR: A rapid on‐site detection of gene doping

Contact Info

Product

Resources

About