High‐Content Screening of Functional Genomic Libraries

Rines, Daniel R.; Tu, Buu P.; Miraglia, Loren; Welch, Genevieve; Zhang, Jia; Hull, Mitchell V.; Orth, Anthony P.; Chanda, Sumit K.

doi:10.1016/s0076-6879(06)14028-8

Cited by 20 publications

(12 citation statements)

References 23 publications

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“…Cre treatment could then liberate master clones containing mutations of interest in a circular form suitable for recovery in bacteria. We also note that there has been significant progress in developing high-throughput screening methods for plant and animal cells (12). Thus, it may prove possible to screen recombination-based mutant libraries in metazoan cells for dominant phenotypic traits.…”

Section: Discussionmentioning

confidence: 99%

“…Advances in functional genomics have opened up the possibility of using genome-wide RNAi or chemical genetics screens to assess null phenotypes for novel genes (12,13). In many cases, however, it would be beneficial to produce and analyze additional types of mutations, either for a more comprehensive genetic analysis or for other types of experiments.…”

Section: Introductionmentioning

confidence: 99%

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One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning

Khalil¹,

Julius²,

Bachant³

2007

Nucleic Acids Research

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Recombination cloning encompasses a set of technologies that transfer gene sequences between vectors through site-specific recombination. Due in part to the instability of linear DNA in bacteria, both the initial capture and subsequent transfer of gene sequences is often performed using purified recombination enzymes. However, we find linear DNAs flanked by loxP sites recombine efficiently in bacteria expressing Cre recombinase and the lambda Gam protein, suggesting Cre/lox recombination of linear substrates can be performed in vivo. As one approach towards exploiting this capability, we describe a method for constructing large (>1 × 106 recombinants) libraries of gene mutations in a format compatible with recombination cloning. In this method, gene sequences are cloned into recombination entry plasmids and whole-plasmid PCR is used to produce mutagenized plasmid amplicons flanked by loxP. The PCR products are converted back into circular plasmids by transforming Cre/Gam-expressing bacteria, after which the mutant libraries are transferred to expression vectors and screened for phenotypes of interest. We further show that linear DNA fragments flanked by loxP repeats can be efficiently recombined into loxP-containing vectors through this same one-step transformation procedure. Thus, the approach reported here could be adapted as general cloning method.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning

Khalil¹,

Julius²,

Bachant³

2007

Nucleic Acids Research

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“…† The use of HCS-as-HTS should be adaptable, not only to other orphan and known GPCRs, but to other target classes such as nuclear transcription factors, where the seminal activation event involves a quantifiable translocation event. Furthermore, the HCS-as-HTS aspect can and has been exploited as a tool for looking at cellular phenotypic changes in siRNA studies, many of which have been large in scale (9)(10)(11). Finally, the use of HCS-as-HTS brings us to the next section of the present work, namely, a large-scale screening campaign using a multiplexed high content cell health/cytotoxicity readout.…”

Section: Hcs For Orphan Gpcrs and Transfluormentioning

confidence: 99%

HCS for HTS

Hoffman

Garippa

2007

High Content Screening

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“…One solution to this problem is the use of relative small pools of B1000 shRNAs in negative selection assays (Ngo et al, 2006;Shaffer et al, 2008). More recently, this approach was optimized as well as used successfully to screen larger shRNA libraries (Rines et al, 2006;Luo et al, 2008;Schlabach et al, 2008;Silva et al, 2008).…”

Section: Loss-of-function Genetic Screening Toolsmentioning

confidence: 99%

“…Such complex phenotypes can be detected using for instance cell sorting or high throughput microscopy. Microscopic images can subsequently be analysed by software that can be programmed to extract features such as cell shape, DNA content, subcellular localization of proteins and other parameters (Wheeler et al, 2005;Pepperkok and Ellenberg, 2006;Rines et al, 2006). This combination of single-well screening and reading of complex phenotypes is referred to as 'high content screening' (Neumann et al, 2006).…”

Section: Loss-of-function Genetic Screening Toolsmentioning

confidence: 99%

Loss-of-function genetic screens as a tool to improve the diagnosis and treatment of cancer

Mullenders

Bernards

2009

Oncogene

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A major impediment to the effective treatment of cancer is the molecular heterogeneity of the disease, which is also reflected in an equally diverse pattern of clinical responses to therapy. Currently, only few drugs are available that can be used safely and effectively to treat cancer. To improve this situation, the development of novel and highly specific targets for therapy is of utmost importance. Possibly even more importantly, we need better tools to predict which patients will respond to specific therapies. Such drug response biomarkers will be instrumental to individualize the therapy of patients having seemingly similar cancers. In this study, we discuss how RNA interference-based genetic screens can be used to address these two pressing needs in the care for cancer patients.

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High‐Content Screening of Functional Genomic Libraries

Cited by 20 publications

References 23 publications

One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning

One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning

HCS for HTS

Loss-of-function genetic screens as a tool to improve the diagnosis and treatment of cancer

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