Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants

Background: For successful in vitro plant regeneration, plant cell lines with multiple transgene integration and low transgene expression levels need to be ruled out. Although real-time polymerase chain reaction (RT-PCR) is a rapid way to accomplish this, it is also expensive and typically limits the size of the target sequence. Quantitative competitive PCR (QC-PCR) is proven to be a safe and accurate method for determination of both copy number and quantification of transcript levels of synthetic transgenes in transformed plants. Results: The glyphosate oxidoreductase genewas chemically synthesized and used to transform Brassica napus L. via Agrobactrium-mediated transformation. A construct containing the mutated form of a synthetic glyphosate oxidoreductase (gox) gene (internal standard) was prepared. Gene copy number was estimated in nine independent transgenic lines using QC-PCR as well as the standard method of Southern blot analysis. By quantitative RT-PCR, transcript levels were also determined in these lines. High (> 3), medium to high (2.2-3), medium to low (1-2.2), and low (< 1) levels of transcript were detected. Conclusions: No direct relationship was found between copy number and transgene expression levels. QC-PCR method could be implemented to screen putative transgenic plants and quickly select single T-DNA inserts. QC-PCR methods and the prepared competitor construct may be useful for future quantification of commercial transgenic food and feed.

High Transcript Level of FLT3 Associated with High Risk of Relapse in Pediatric Acute Myeloid Leukemia

Identification of prognostic factors and risk-based post-remission therapy was proposed to improve the outcomes of acute myeloid leukemia (AML) and a mutation of FLT3 has been reported to be a risk factor, especially for pediatric patients. Recently, FLT3 expression level was implicated to have prognostic significance in adults, but little is known for childhood AML. To define the prognostic significance, transcript level of FLT3 was analyzed in 52 pediatric AML patients. The median copy number of FLT3 was 4.6x10(3) (40-5.9x10(7) copies)/1.0x10(6) GAPDH copy, and the relapse free survival of patients with high transcript level of FLT3 (>10(6) copy number) (0%) was significantly lower than that of the others (53.2%). High transcript level of FLT3 was associated with a markedly high risk of relapse. The development of new therapeutic scheme such as a frontline allogeneic stem cell transplantation or administration of FLT3 inhibitor is needed to improve outcomes.