Arrayed adenoviral expression libraries for functional screening.

With the publication of the sequence of the human genome, we are challenged to identify the functions of an estimated 70,000 human genes and the much larger number of proteins encoded by these genes. Of particular interest is the identification of gene products that play a role in human disease pathways, as these proteins include potential new targets that may lead to improved therapeutic strategies. This requires the direct measurement of gene function on a genomic scale in cell-based, functional assays. We have constructed and validated an individually arrayed, replication-defective adenoviral library harboring human cDNAs, termed PhenoSelect library. The adenoviral vector guarantees efficient transduction of diverse cell types, including primary cells. The arrayed format allows screening of this library in a variety of cellular assays in search for gene(s) that, by overexpression, induce a particular disease-related phenotype. The great majority of phenotypic assays, including morphological assays, can be screened with arrayed libraries. In contrast, pooled-library approaches often rely on phenotype-based isolation or selection of single cells by employing a flow cytometer or screening for cell survival. An arrayed placental PhenoSelect library was screened in cellular assays aimed at identifying regulators of osteogenesis, metastasis, and angiogenesis. This resulted in the identification of known regulators, as well as novel sequences that encode proteins hitherto not known to play a role in these pathways. These results establish the value of the PhenoSelect platform, in combination with cellular screens, for gene function discovery.

Improved lipid profile through liver-specific knockdown of liver X receptor alpha in KKAy diabetic mice.

Nuclear hormone receptors liver X receptor (LXRalpha and LXRbeta) ligands are attractive approaches for the treatment of dyslipidemia and atherosclerosis. To further elucidate the function of LXRalpha in liver lipid metabolism in a disease-relevant animal model, the KKAy mouse, we used adenoviral vectors to selectively knock down LXRalpha gene expression. Out of five different short hairpin RNAs (shRNAs) that were tested in vitro, one construct was selected for detailed analysis of LXRalpha knockdown in vivo. Reduction of LXRalpha transcript levels to 48 +/- 13% compared with control virus transduction resulted in a significant downregulation of the LXRalpha-regulated lipogenic genes sterol-regulatory element binding protein-1c (SREBP1c) and stearoyl CoA desaturase 1 in vivo. Interestingly, ABCA1 and phoshoenolpyruvate carboxykinase 1 expression was not affected, whereas lipoprotein lipase (LPL) expression was found to be increased. In addition, 8 days after virus transduction, both plasma and liver triglycerides (TGs) were reduced by about 50%. Changes in TG levels were not due to reduced food intake in virus-treated animals, because pair-fed mice showed unchanged TG levels. Taken together, liver-specific knockdown of LXRalpha in vivo by shRNA reduced expression of lipogenic master genes, like SREBP1c, and improved the lipid profile of hypertriglyceridemic KKAy mice.

Adenoviral vectors expressing siRNAs for discovery and validation of gene function.

RNA interference is a powerful tool for studying gene function and for drug target discovery in diverse organisms and cell types. In mammalian systems, small interfering RNAs (siRNAs), or DNA plasmids expressing these siRNAs, have been used to down-modulate gene expression. However, inefficient transfection protocols, in particular, for primary cell types, have hampered the use of these tools in disease-relevant cellular assays. To be able to use this technology for genome-wide function screening, a more robust transduction protocol, resulting in a longer duration of the knock-down effect, is required. Here, we describe the validation of adenoviral vectors that express hairpin RNAs that are further processed to siRNAs. Infection of cell lines, or primary human cells, with these viruses leads to an efficient, sequence-specific, and prolonged reduction of the corresponding target mRNA, resulting in a reduction of the encoded protein level in the cell. For knock-down of one of the targets, GalphaS, we have measured inhibition of ligand-dependent, G-protein-coupled signaling. It is expected that this technology will prove to be of great value in target validation and target discovery efforts.