Large Scale Virus Vector Based Libraries

One major criticism of cell-based screening in general is that the cell lines normally used to measure gene function are too far removed from physiologically relevant cell types such as those derived directly from primary tissue. This is partly due to the difficulty of obtaining a primary cell type with the longevity in culture required for engineering, if necessary, or for the screen itself, and due to the difficulty of delivering genetic material to these cell types. Most applications involve transient delivery of genetic material using lipid-based transfection reagents to which many primary cell types are resistant. To overcome this obstacle for more physiologically relevant screens, vector-based RNA interference libraries have been constructed in recombinant retroviral vectors.171'172 Retroviruses afford the ability to introduce transgenes via stable infection to a broad range of primary tissue types (and cell lines) with high efficiency. For example, Berns et al. synthesized shRNA encoding retroviruses against 7914 human genes (NKi library) involved in major cellular pathways, including cell cycle, transcription regulation, stress signaling, signal transduction, and other important biological processes and human diseases.171 Validation of the library was demonstrated by screening for modulators of p53-dependent proliferation arrest, resulting in successful identification of five new genes involved in the p53 tumor suppressor pathway and p53 itself. Concurrently, Paddison et al. reported an shRNA library covering more than 9600 human and 5500 mouse genes.172 The library was designed with a retroviral vector pSHAG MAGIC that contains the hairpin sequence under the control of the U6 RNA polymerase III promoter and homologous recombination sites that allow easy shuttling between different vector backbones by a bacterial mating system. Paddison et al.have provided validation for the library by identifying components of the proteasome complex using a modified green fluorescent protein that is degraded by the proteasome, confirming 15 known proteasome subunits. Recently, a second-generation library comprising 62000 constructs covering over 28000 human genes, based on the library used by Paddison et al., developed in collaboration with Hannon Elledge was made commercially available from Open Biosystems. Using this resource, Westbrook et al. were able to identify genes that suppress oncogenic transformation in a human mammary epithelial cells (HMECs).191 Immortalized HMECs expressing naturally high levels of Myc exhibit a proliferation arrest in absence of extracellular matrix proteins. Knockdown of genes leading to anchorage-independent growth were assessed. Two established tumor suppressors, TGFBR2 and PTEN, were identified as was REST/NRSF, a transcriptional repressor of neuronal gene expression, a potential new candidate tumor suppressor gene. Kolfschoten et al. utilized the library of Bernards and colleagues in a screen for novel tumor-suppressor genes which, when silenced, substituted for the activity of oncogenic RAS. This screen led to the identification of PITX1 as a promoter of anchorage-independent growth in fibroblasts.

As a result of the power of retroviral-based genetic screens, several groups have combined their efforts to expand this resource. The RNAi consortium (TRC), a collaborative effort among six research institutions and five international life sciences organizations, released a lentiviral shRNA library consisting of approximately 35 000 shRNA constructs targeting 5300 human and 2200 mouse genes. The first-generation library named MISSION TRC-Hs 1.0 (Sigma-Aldrich) or Expression Arrest TRC (Open Biosystems) is based on the self-inactivating lentiviral vector pLKO.1, wherein expression of the hairpin sequence is driven by the U6 promoter.193 The effectiveness of this newly released lentiviral library has not been reported; however, the results of the aforementioned screens indicate the potential utility of this resource.

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