Peter Herwig Maria Tomme

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.

Rapid determination of adenoviral vector titers by quantitative real-time PCR

Replication defective adenoviruses have been used as vectors in a variety of settings including gene transfer, gene manipulation, and functionality studies. A quantitative real-time PCR-based assay is described for rapid determination of physical titers of recombinant adenovirus vectors. This method is based on amplification of a 77 bp fragment located near the left end of the adenovirus type 5 genome. Evaluation of this method demonstrated that it is simple, sensitive and reproducible, and has a dynamic range of quantitation over 5 logs. This assay is applicable to purified adenovirus as well as vectors prepared by simple cell lysis procedure, requiring only a small amount of starting material. The simplicity and short turn-around time of this assay should facilitate rapid titer determination for a large collection of adenoviral vectors.