Genetic dissection of signaling pathways in human disease is the current challenge in drug target identification. The ability to generate libraries of cDNAs from human tissue sources and clone into mammalian expression vectors is enabling this field. Harnessing viruses to introduce libraries blan-ketly into cells, paired with advances in human tissue culture systems and phenotypic readouts have ushered in the age of human cell genetics. In the late 1980s, Brian Seed established the expression screening approach in mammalian cells and laid the groundwork for functional gene identification in cell systems relevant to human physiology (Allen and Seed, 1989). In the technology's infancy, functional cDNA expression screening identified genes such as LFG, an antiapoptotic gene, Toso, a T-cell surface receptor that blocks FAS-induced apoptosis, multiple members of the JAK and STAT interferon signaling cascade, and NF-kB activating genes such as IKK-gamma (Darnell et al., 1994; Hitoshi et al., 1998; Yamaoka et al., 1998). These screens were done by ectopic overexpression of pooled cDNAs, looking for genes which confer "gain of function." Despite obvious utility, this pooled screening strategy is analogous to "finding a needle in a haystack"; first the chance of finding something in the haystack is very slim based on the abundance of the gene of interest, and second, one is not even assured the gene of interest is in the proverbial stack in the first place. More precisely, the limitations include: (1) strong dependence on an assay's signal-to-noise ratio; (2) nonnormalized libraries make rare, low abundance genes more difficult to find; (3) libraries usually tissue specific and lack many genes; (4) are ridden with cDNA fragment; and (5) rescuing the causal cDNA from the cell of interest can be difficult.
As a result of the completed human genome sequence, an effort to clone all human genes, in expression-ready full-length form are becoming available to facilitate genome-wide functional screens in mammalian cells. This new approach circumvents many of the problems associated with pool screening. Full-length cDNAs, encoded by mammalian expression vectors, including both known and predicted genes are arrayed in individual wells of microtiter plates, and systematically introduced in parallel into reporter cells.
These libraries can include the 12,000 plus human genes assembled by the Mammalian Genome Collection (http://mgc.nci.nih.gov/), the 24,000 unique member Origene collection (http://www.origene.com/cdna/), all of which exist in mammalian expression ready vectors for high-level expression via transient transfection or in virus-based vectors for screens in primary cells (Gerhard et al., 2004; Strausberg et al., 2002). The advantages of arrayed-based formats over pooled-library approaches are: (1) the reporter assays do not require a very high signal-to-noise ratio, since each gene is tested individually for function; (2) the libraries are normalized such that rare genes have an equal opportunity to register as hits; and (3) rescuing the causal gene is unnecessary since the identity of each gene in each well is already known. Another advantage soon to be realized from a comprehensive set of all human genes arrayed for functional screening is that one will no longer have to wonder if the gene of interest resides in the library if no hits score as positives—in this case, perhaps a gene with the desired function does not exist.
The arrayed functional screening approach is beginning to offer new targets in oncology. This gain-of-function screening approach has particular utility in oncology screens given that the etiology of many cancers stems from misregulation and overexpression of oncogenes. Chanda et al. (2003) reported a strategy to identify positive regulatory effectors of AP-1-dependent mitogenesis using an AP-1-responsive reporter vector upstream of a reporter gene encoding. Michiels et al. (2002) used a 13,000 member-arrayed adenoviral expression library to identify novel regulators of osteo-genesis, metastasis, and angiogenesis. Iourgenko et al. (2003) used a similar methodology to identify genes which activate expression of interleukin-8, a cytokine with etiological bases in asthma, arthritis, and cancer. Other reports utilize various reporter constructs in genome-wide functional analysis to analyze NF-kB signaling pathways, to identify p53 regulators or to identify new components of the Wnt signaling pathway, an important controller of embryonic development and possibly cancer (Huang et al., 2004; Liu et al., 2005; Matsuda et al., 2003). The main advantage of the arrayed approach is that one typically discovers more genes than from the pooled approach, which serves to increase the probability of finding one that satisfies "good target" criteria.
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