Dataset Information

Transgene detection by digital droplet PCR.

ABSTRACT: Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.

SUBMITTER: Moser DA

PROVIDER: S-EPMC4222945 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Transgene detection by digital droplet PCR.

Moser Dirk A DA Braga Luca L Raso Andrea A Zacchigna Serena S Giacca Mauro M Simon Perikles P

PloS one 20141106 11

Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PC ...[more]

PMID: 25375130

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Project description:BackgroundCell transplantation is in clinical development for the treatment of various ailments including acquired and inborn hepatic diseases. Detection and quantification of the donor cells after infusion remain difficult. Traditional methods (sex-based FISH, HLA mismatch, and Short Tandem Repeat PCR) can only achieve low levels of sensitivity (1%) and therefore are seldom used. The use of a droplet digital PCR (ddPCR) assay based on mismatch of null alleles is a promising alternative.MethodsWe selected genes with a high frequency of null genotype in the general population (SRY, RHD, TRY6, LEC3C, GSTM1, and GSTT1) and investigated their expression by liver progenitor cell donors and liver cell therapy recipients, in order to identify genes of interest for each donor/recipient couple. We first validated the detection of microchimerism by ddPCR and then used these assays to detect and quantify microchimerism in pre- and postinfusion liver biopsies.ResultsWe validated the ddPCR detection of the selected genes based on linearity, precision, lack of inhibition, and accuracy, and we established limits of blank, limits of detection, and limits of quantification to ensure the reliability of the results. After genotyping donors and recipients, we were able to identify at least one gene of interest for each donor/recipient couple. We detected donor cells in the three patients posttransplantation. However, analysis of several biopsies taken at the same timepoint revealed a heterogeneous cell distribution. In addition, the values obtained remained below the limit of quantification. Therefore, the actual quantification of microchimerism may not be entirely accurate.ConclusionsOverall, our study demonstrates that the detection of microchimerism post-liver cell transplantation can be performed using ddPCR amplification of null allele genes expressed by the donor but absent from the recipient. However, this technique can be extended to other cell types and target organs in cell transplantation.

Dataset Information

Transgene detection by digital droplet PCR.

Publications

Transgene detection by digital droplet PCR.

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