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The incidence of congenital cardiovascular malformations is approximately 1 percent of live births in the United States and includes a spectrum of structural and functional defects in heart development. It is estimated that an additional 1 percent to 2 percent of the population has congenital malformations that manifest later in life as adult cardiovascular disease.
In many cases, the underlying cause of these congenital malformations is aberrant signal transduction or altered function of downstream effectors. Examples of signaling mechanisms associated with congenital heart malformations are TGFbeta with Marfan syndrome, Shp2 with Noonan syndrome and Notch1 with bicuspid aortic valve. In addition to causing congenital cardiac malformations, each of these signaling pathways also contributes to cardiovascular disease after birth. Therefore it is of critical importance to identify and characterize all of the molecular constituents and downstream targets of these regulatory networks in heart development and disease.
The goal is to define the critical regulatory components in multiple signaling pathways that cause congenital heart malformations and subsequent cardiovascular disease and to make these candidates available to the scientific community. We expect that these studies will lead to the discovery of new therapeutic targets for the treatment of congenital cardiovascular disease to be shared.
To carry out an unbiased screen for interacting partners in this disease pathway, we have, in collaboration with the Howard Hughes Medical Institute, developed a robotic screening facility capable of robustly assaying entire genomes for sequences that might interact with the pathway under study.
The technology underlying the proposed mammalian cell-based screen is that developed by the RNAi Consortium. These investigators generated a genome-wide library of shRNAs (both human and mouse) contained within individual lentiviruses specifically designed for high-throughput screening (HTS). The mouse library contains multiple clones (each clone represents a unique sequence for mRNA knockdown) for individual targets with 99.6 percent of genes having at least four clones, and high-value genes having five or more clones. The entire clone set for the mouse shRNA library now consists of 77,713 separate plasmids. These are contained in 820 individual 96-well plates as frozen bacterial stocks organized in a bar-coded rack system. We have recently purchased the entire library as plasmid stocks (each representing an individual clone), as well all the necessary automated systems (see description of equipment) for high-throughput production of lentivirus.
The benefit of the lentiviral system over other expression systems, such as transfection, is multifold:
We are currently using this HTS technology to look for interacting partners that affect cell signaling, protein misfolding and aggregation-prone physiological stress pathways in the cardiomyocyte.
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Shp2 and signaling through ERK. After ligand binding, the RTK homodimerizes and autophosphorylates, creating an SH2 binding domain, at which a multiprotein “signalosome” assembles. Activated Shp2 results in activation of Ras through an as yet undefined process, and activated Ras, in turn, recruits Raf, a serine-threonine kinase, to the membrane. Shp2 gain-of-function (GOF) results in prolongation of active Ras signaling through the ERK branch of the MAPK pathway. Many potential partners and proteins may impact on the signaling processes that are outlined.
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