Development and Optimization of a High Titer Recombinant Lentivirus System.

To enable simple and effective high titer recombinant lentivirus production, we examined key parameters for the generation of lentivirus including: transfection optimization, media change, incubation time and DNA vector selection. These results illustrate the importance of optimizing transfection processes for high titer recombinant lentivirus production.

A facile method for simultaneously measuring neuronal cell viability and neurite outgrowth.

Neurite outgrowth is an important morphological phenotype of neuronal cells that correlates with their function and cell health, yet there are limited methods available for measuring this phenomenon. Current approaches to measuring neurite outgrowth are laborious and time-consuming, relying largely upon immunocytochemical staining of neuronal markers (e.g., beta-III tubulin or MAP2) followed by manual or automated microscopy for image acquisition and analysis. Here we report the development of a quick and simple dual-color fluorescent dye-based staining method that allows for the simultaneous measurement of neuronal cell health and relative neurite outgrowth from the same sample. An orangered fluorescent dye that stains cell membrane surfaces is used as an indirect reporter of changes in relative neurite outgrowth due to alterations in the number or length of membrane projections emanating from neuronal cell bodies. Cell viability is assessed simultaneously via the use of a cell-permeant dye that is converted by intracellular esterase activity from a non-fluorescent substrate to a green-fluorescent product. Using Neuroscreen-1 cells (a PC-12 subclone), primary rat cortex neurons, and human induced pluripotent stem cell (iPSC)-derived neurons, we demonstrate that this multiplex assay allows for rapid visualization and unbiased, quantitative plate reader analysis of neuronal cell health and neurite outgrowth.

A homogeneous fluorescent live-cell assay for measuring 7-transmembrane receptor activity and agonist functional selectivity through beta-arrestin recruitment.

Seven-transmembrane (7TM) receptors play an essential role in the regulation of a wide variety of physiological processes, making them one of the top target classes for pharmaceuticals. 7TM receptor function is mediated and modulated through 2 primary processes: G-protein and beta-arrestin signaling. Classically, it has been recognized that these 2 processes can interact with one another during 7TM receptor desensitization, but it has more recently been recognized that these 2 processes can also act independently of one another and can activate parallel signaling pathways. As such, the methods used to interrogate 7TM receptor signaling, both from a biological and a pharmaceutical perspective, may need to be reevaluated and the question of whether functionally selective compounds (compounds that selectively activate one pathway over another) can be rationally developed must be raised. Although numerous high-throughput screening (HTS) compatible assays exist for studying second messengers arising from G-protein signaling, far fewer HTS compatible assays exist for studying beta-arrestin recruitment. The authors report on the Tango 7TM receptor assay technology, a high-throughput homogeneous assay method for monitoring beta-arrestin recruitment that uses a live-cell fluorescent readout. This assay format is broadly applicable to 7TM receptors, independent of G-protein coupling and, as such, has been used to produce assays for over 70 7TM receptor targets. The authors also show how flow cytometry can be used to select clones with desired pharmacological profiles and how an inducible expression system can increase the assay window for targets with high levels of constitutive activity. Finally, they demonstrate how the Tango system can be used in parallel with assays aimed at second-messenger signaling to enable functional selectivity studies.

High-throughput screening compatible cell-based assay for interrogating activated notch signaling.

Aberrant Notch pathway function is associated with a wide array of developmental disorders, cancers, and neurodegenerative diseases. Thus, strategies to modulate Notch signaling may facilitate therapeutic intervention. Ligand binding to Notch receptors at the cell surface results in a series of cleavage events that release Notch intracellular domain (NICD) fragments that translocate to the nucleus where they function as transcriptional activators of downstream transcriptional programs. We have developed a cell-based assay that can be used to screen for modulators of NICD signaling by engineering HeLa cervical cancer cells with a Notch response element driving beta-lactamase (BLA) reporter gene expression along with a tetracycline-inducible NICD expression system. Induction of NICD expression leads to increased BLA reporter activity that can be knocked down using NICD-specific RNA interference as well as RNA interference against endogenous components of the NICD transactivation complex. Profiling of 19 known compounds in this assay identified several previously undescribed modulators of NICD signaling. The Wnt pathway inhibitor ICG-001 antagonized NICD signaling, whereas the histone deacetylase inhibitor suberoylanilide hydroxamic acid, the heat shock protein 90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin, and the dual phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin inhibitor PI-103 each further activated the NICD-driven reporter activity. The AKT inhibitor triciribine and the PI3K inhibitor GDC-0941 also resulted in the enhanced reporter activity, strongly implicating a role for the PI3K/AKT pathway in regulating NICD signaling. Together this cell-based assay system provides a sensitive, quantitative readout for NICD signaling that is amenable to high-throughput screening for NICD pathway modulators.

Pharmacological characterization of purified recombinant mTOR FRB-kinase domain using fluorescence-based assays.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in nutrient sensing and cell growth and is a validated target for oncology and immunosuppression. Two modes of direct small-molecule inhibition of mTOR activity are known: targeting of the kinase active site and a unique mode in which the small molecule rapamycin, in complex with FKBP12 (the 12-kDa FK506 binding protein), binds to the FRB (FKBP12/rapamycin binding) domain of mTOR and inhibits kinase activity through a poorly defined mechanism. To facilitate the study of these processes, the authors have expressed and purified a truncated version of mTOR that contains the FRB and kinase domains and have developed homogeneous fluorescence-based assays to study mTOR activity. They demonstrate the utility of these assays in studies of active site-directed and FRB domain-directed mTOR inhibition. The results suggest that these assays can replace traditional radiometric or Western blot-based assays.