TAG-RNAi overcomes off-target effects in cancer models.

RNA interference offers therapeutic opportunities for the clinical targeting of otherwise undruggable oncogenes. However RNAi can have off-target effects that considerably increase treatment risks. To manage these side effects and allow an easy subtraction of their activity in healthy tissues, we present here the TAG-RNAi approach where cells that are not designated targets do not have the mRNA tag. Using TAG-RNAi we first established the off-target signatures of three different siRNAs specific to the Cyclin D1 oncogene by RNA-sequencing of cultured cancer cells expressing a FLAG-HA-tagged-Cyclin D1. Then, by symmetrical allografts of tagged-cancer cells and untagged controls on the left and right flanks of model mice, we demonstrate that TAG-RNAi is a reliable approach to study the functional impact of any oncogene without off-target bias. Finally we show, as examples, that mutation-specific TAG-RNAi can be applied to downregulate two oncogenic mutants, KRAS-G12V or BRAF-V600E, while sparing the expression of the wild-type proteins. TAG-RNAi will thus avoid the traditional off-target limitations of RNAi in future experimental approaches.

Hyper sensitive protein detection by Tandem-HTRF reveals Cyclin D1 dynamics in adult mouse.

We present here a novel method for the semi-quantitative detection of low abundance proteins in solution that is both fast and simple. It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target. This novel approach we termed "Tandem-HTRF", can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format. Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge. Thus our method, Tandem-HTRF offers a promising way to investigate subtle variations in the dynamics of sparse proteins from limited biological material.

Genome-wide profiling of G protein-coupled receptors in cerebellar granule neurons using high-throughput, real-time PCR.

BACKGROUND: G protein-coupled receptors (GPCRs) are major players in cell communication, regulate a whole range of physiological functions during development and throughout adult life, are affected in numerous pathological situations, and constitute so far the largest class of drugable targets for human diseases. The corresponding genes are usually expressed at low levels, making accurate, genome-wide quantification of their expression levels a challenging task using microarrays. RESULTS: We first draw an inventory of all endo-GPCRs encoded in the murine genome. To profile GPCRs genome-wide accurately, sensitively, comprehensively, and cost-effectively, we designed and validated a collection of primers that we used in quantitative RT-PCR experiments. We experimentally validated a statistical approach to analyze genome-wide, real-time PCR data. To illustrate the usefulness of this approach, we determined the repertoire of GPCRs expressed in cerebellar granule neurons and neuroblasts during postnatal development. CONCLUSIONS: We identified tens of GPCRs that were not detected previously in this cell type; these GPCRs represent novel candidate players in the development and survival of cerebellar granule neurons. The sequences of primers used in this study are freely available to those interested in quantifying GPCR expression comprehensively.

The small GTPase RhoV is an essential regulator of neural crest induction in Xenopus.

In vertebrates, the Rho family of GTPases is made of 20 members which regulate a variety of cellular functions, including actin cytoskeleton dynamics, cell adhesion and motility, cell growth and survival, gene transcription and membrane trafficking. To get a comprehensive view of Rho implication in physiological epithelial-mesenchymal transition, we carried out an in situ hybridization-based screen to identify Rho members expressed in Xenopus neural crest cells, in which we previously reported RhoB expression at the migrating stage. In the present study, we identify RhoV as an early expressed neural crest marker and provide evidence that its activity is essential for neural crest cell induction. RhoV mRNA is maternally expressed and accumulates shortly after gastrulation in the neural crest forming region. Using antisense morpholino injection, we show that at neurula stages, RhoV depletion impairs expression of the neural crest markers Sox9, Slug or Twist but has no effect on Snail induction. At the tailbud stage, RhoV knockdown causes a dramatic loss of cranial neural crest derived structures. All these defects are rescued by ectopic wild-type RhoV, whose overexpression on its own expands the neural crest territory. Our findings disclose an unprecedented Rho function in pathways that control neural crest cells specification.