An accurate, reliable, and universal qPCR method to identify homozygous single insert T-DNA with the example of transgenic rice.

Early determination of transgenic plants that are homozygous for a single locus T-DNA insert is highly desirable in most fundamental and applied transgenic research. This study aimed to build on an accurate, rapid, and reliable quantitative real-time PCR (qPCR) method to fast-track the development of multiple homozygous transgenic rice lines in the T1 generation, with low copy number to single T-DNA insert for further analyses. Here, a well-established qPCR protocol, based on the OsSBE4 reference gene and the nos terminator, was optimized in the transgenic Japonica rice cultivar Nipponbare, to distinguish homozygous single-insert plants with 100% accuracy. This method was successfully adapted to transgenic Indica rice plants carrying three different T-DNAs, without any modifications to quickly develop homozygous rice plants in the T1 generation. The accuracy of this qPCR method when applied to transgenic Indica rice approached 100% in 12 putative transgenic lines. Moreover, this protocol also successfully detected homozygous single-locus T-DNA transgenic rice plants with two-transgene T-DNAs, a feature likely to become more popular in future transgenic research. The assay was developed utilizing universal primers targeting common sequence elements of gene cassettes (the nos terminator). This assay could therefore be applied to other transgenic plants carrying the nos terminator. All procedures described here use standardized qPCR reaction conditions and relatively inexpensive dyes, such as SYBR Green, thus the qPCR method could be cost-effective and suitable for lower budget laboratories that are involved in rice transgenic research.

The dawn of targeted therapies for triple negative breast cancer (TNBC): a snapshot of investigational drugs in phase I and II trials.

INTRODUCTION: Triple negative breast cancer (TNBC) was once thought to be an insurmountable disease marked by a lack of targeted treatments. However, we are now witnessing the dawn of targeted therapies for TNBC in which progress has stemmed from an improved understanding of the components that make TNBC unique. The identification of biomarkers, such as BRCA1/2, PIK3CA and RSK2, have advanced the field remarkably and there is considerable interest in finding novel therapeutics for TNBC that offer durable clinical benefit with fewer adverse events. AREAS COVERED: We discuss phase I/II trials of new and emerging targeted therapies for TNBC, according to ClinicalTrials.gov up to June 2020. Although the emphasis is on ongoing and completed early phase trials, we also highlight pivotal studies that have led to the approval of new targeted classes of drugs for TNBC, with a focus on outcomes and common adverse events of each class of therapy. EXPERT OPINION: The way forward for TNBC treatment is through precision medicine. The use of novel agents matched with biomarkers to identify patients with the best chance of sustainable response offers new hope. We now have great potential for improving the outcomes for patients with TNBC.