Arrayed adenoviral expression libraries for functional screening

Abstract

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.

Substrate-mediated delivery from self-assembled monolayers: Effect of surface ionization, hydrophilicity, and patterning

“…Transfected cell arrays have been formed using a substrate-mediated approach in which plasmids or adenoviruses were mixed with collagen and spotted onto glass slides or into wells [46][47][48]. Plated cells were transfected and could be analyzed for cellular responses using a variety of imaging or biochemical techniques.…”

“…Also, DNA complexes have been mixed with fibronectin and spotted onto glass slides for arrayed transfection of human mesenchymal stem cells [51]. While transfected cell arrays hold great potential [46][47][48][49][50][51], further development of a substratemediated delivery system that efficiently transfects a wide variety of primary cells and cell lines, while allowing for spatially-controlled delivery within the different domains is required [46,52,53]. Using SAMs on gold to form transfected cell arrays can allow precise control over surface chemistries, interactions between the substrate and DNA complexes, transfection efficiencies, and pattern sizes to create a well-characterized and efficient array delivery system.…”

shRNA libraries and their use in cancer genetics

“…Such vectors provide a renewable source of a gene-silencing reagent that can mediate persistent gene silencing after stable integration of the vector into the host-cell genome. Furthermore, the core silencing 'hairpin' cassette can be readily inserted into retroviral, lentiviral or adenoviral vectors, facilitating delivery of shRNAs into a broad range of cell types [4][5][6][7][8] . Additonally, regulatable versions of shRNA vectors have been generated and used successfully in genetic screens 9,10 .…”

Robust functional gene validation by adenoviral vectors: one-step Escherichia coli-Derived Recombinant Adenoviral Genome construction

“…Many approaches have been described for generating recombinant adenoviruses (rAV), principally involving direct plasmid construction of recombinant adenoviral genomes, [4][5][6][7][8] or indirect construction (two-plasmid systems). 3,[9][10][11][12][13][14][15][16] In general, one-plasmid systems are limited by choice of suitable restriction sites and the difficulties encountered in manipulating such large plasmids (B50 kbp). Two-plasmid systems include approaches whereby a shuttle plasmid (or DNA fragment), containing an expression cassette, is combined with a second (larger) plasmid (or viral DNA fragment) containing the majority of the adenoviral coding regions by homologous recombination in Escherichia coli, 9-11 or in mammalian packaging cell lines.…”

“…Two-plasmid systems include approaches whereby a shuttle plasmid (or DNA fragment), containing an expression cassette, is combined with a second (larger) plasmid (or viral DNA fragment) containing the majority of the adenoviral coding regions by homologous recombination in Escherichia coli, 9-11 or in mammalian packaging cell lines. 3,[12][13][14][15] Cre-lox-mediated rAV plasmid assembly has also been described, in both mammalian cell lines 13 and E. coli. 16 Additionally, methods for construction of adenoviral genomes by homologous recombination in yeast and their subsequent modification by two-step gene replacement have been described.…”