Complex assay tools such as reporter based cis and trans activation systems are being developed for screening several therapeutic targets. Further, we are developing a panel of reporter based functional assays to simultaneously monitor key transcription factors downstream of receptor activated signaling pathways. We will use this panel to simultaneously profile the activity of a panel of 30+ GPCRs as well as other receptors to better understand cellular cross-talk and predict side effects. This system can also help identify the molecular mechanism of action of NCEs for which cell based or in vivo activity has been established. The yeast group creates human disease models by knocking out native yeast genes using homologous recombination and complementing the phenotype with human homologues. Yeast is also used for other specialized applications such as two hybrid screens. One new thrust area is the development of a yeast growth assay with genetically modified strains to assay those human targets that have been refractory to HTS based approaches.
Our gene expression team amplifies genes from a large panel of human lines and clones them into vectors appropriate for specific applications. We use a variety of host systems for expression - bacterial, yeast, insect and mammalian.

SPECIAL MOLECULAR TOOLS - Reporter based transactivation for Nuclear Hormone Receptors and key Signal Transduction Nodes, RNA interference

Trans-activation and mammalian cell-based reporter gene screen for human nuclear hormone receptors

PPAR α,δ1,δ2, γ
Estrogen α,β
Androgen
Progesterone
Vitamin D
Glucocorticoid
Thyroid hormone α1,α2, β
Mineralocorticoid

Functional networks - key Signal Transduction Nodes

C-JUN
c-FOS
CREB
CHOP
ATF2
ELK1
STAT1

cis- activation reporter gene screen for human transcription factors

NFAT
AP1
NFkB
n-MYC
GAS
Pokemon

RNA Interference

In vitro assays against Dicer and Argonaute-2
Cell based RNAi

YEAST BASED MODELS - HTS, disease models, two hybrid and special screens

HTS for mammalian enzymes

Human Topoisomerases
Human p38 MAP kinase
Human PARP1
Specific human potassium channel for CNS

Yeast based human disease models

Genetically engineered yeast strains can sometime serve as human disease models. Knocking out endogenous homologues of human targets and complementing them with desired human genes creates easily manipulated strains for functional screens. We have developed a model for human Myotubular myopathy (hMTM-1) as an example and can do the same for other genes.

Specialized yeast tools

Yeast 2-hybrid system
Execution of any type of 2-hybrid analysis and subsequent confirmation to identify proteins that interact with a novel target

Free radical detection system
Monitoring the level of SOD1 and SOD2 in complemented KO strains

AMPLIFICATION, CLONING AND EXPRESSION

PCR based amplification, cloning, sequence verification and expression of genes
Custom cDNA library preparation
Panel of 40+ human cell lines to rapidly source RNA
Macro-array for RNA distribution analysis
E.coli, yeast, insect cell and mammalian expression
Stable mammalian cell line generation
Recombinant protein production