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Results for “phenotypic screening”

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PubMed1999

Identifying clinically important gram-negative anaerobes from the oral cavity.

Gram-negative oral anaerobes have frequently been associated with periodontal disease, some species more frequently than others. The confusing classification of these organisms has often obscured the association with disease of particular species within this group of organisms. This investigation aimed to compare different identification methods that could be applied in clinical research. Clinical isolates were collected and identified by three different methods: screening with phenotypic tests, commercial identification kits, and a 16S rRNA-based polymerase chain reaction (PCR) method. Forty-three reference strains of 19 Prevotella and Porphyromonas species were also included in the investigation. The phenotypic screen easily differentiated Porph. gingivalis from the other pigmented species. The screen also gave a good indication of separation of the lactose-fermenting species from the lactose non-fermenting species, although diversity can be seen in beta-galactosidase activity. Commercial identification kits did not add much to identification achieved with the phenotypic screen, only 20% of Porph. gingivalis isolates could be identified to species level with the kits. Neither the kits nor the phenotypic screen could differentiate Pr. intermedia and Pr. nigrescens. With the PCR method, Pr. intermedia and Pr. nigrescens were easily separated, and Porph. gingivalis was readily identified. Because of 16S rRNA gene sequence similarity, Pr. melaninogenica and Pr. veroralis could not be separated by the PCR method.

Bacterial Typing Techniques↗
PubMed2004

Functional screening of drug target genes: m1 muscarinic acetylcholine receptor phenotypes in degenerative dementias.

BACKGROUND AND OBJECTIVES: A number of recent studies surveying single nucleotide polymorphisms within the exonic regions of human genes have revealed a significant number of such variants, including many non-synonymous variants. This highlights the need to directly identify, within individual clinically well-defined patients, those variants that alter protein function as well as structure. We report on the development of a novel phenotypic screening process that combines high-throughput molecular cloning techniques with functional expression utilizing the cell-based assay R-SAT. METHODS: We applied the phenotypic screening process to an analysis of the m1 muscarinic acetylcholine receptor (CHRM1) gene in a cohort of 74 individuals, including 48 diagnosed with neurodegenerative disease, primarily Alzheimer disease, who have been stratified according to their clinical response to the acetylcholinesterase inhibitor donepezil. Phenotypic screening of the CHRM1 gene involved PCR-based amplification from genomic DNA and heterologous expression in mammalian cells. RESULTS: Phenotypic screening yielded functional responses to the agonist carbachol displaying a mean potency (-pEC(50)+/- standard deviation) of 5.8 +/- 0.2, which did not differ from that observed with expression of the wild-type receptor gene (6.0 +/- 0.3). No altered levels of constitutive receptor activity were observed. Dideoxy sequencing did not reveal any non-synonymous variants in the coding exon of this gene within this clinical cohort, while detecting three synonymous variants. CONCLUSION: The results confirm that the m1 receptor gene (CHRM1) is not highly polymorphic in the human population, suggesting that genetic variation within the coding exon of this gene is not a contributing factor to the clinical variability observed during treatment of dementia with cholinergic enhancement therapies.

Aged↗
PubMed2006

Uncovering the uncharacterized and unexpected: unbiased phenotype-driven screens in the mouse.

Phenotype-based chemical mutagenesis screens for mouse mutations have undergone a transformation in the past five years from a potential approach to a practical tool. This change has been driven by the relative ease of identifying causative mutations now that the complete genome sequence is available. These unbiased screens make it possible to identify genes, gene functions and processes that are uniquely important to mammals. In addition, because chemical mutagenesis generally induces point mutations, these alleles often uncover previously unappreciated functions of known proteins. Here we provide examples of the success stories from forward genetic screens, emphasizing the examples that illustrate the discovery of mammalian-specific processes that could not be discovered in other model organisms. As the efficiency of sequencing and mutation detection continues to improve, it is likely that forward genetic screens will provide an even more important part of the repertoire of mouse genetics in the future.

Alleles↗
PubMed2002

Benefit of population-based screening for phenotypic hemochromatosis in young men.

BACKGROUND: Hereditary hemochromatosis (HH) is a common genetic disease leading to accumulation of iron in the body, most notably in the liver. More men than women become clinically ill. The prognosis is excellent if phlebotomy treatment is started before liver cirrhosis develops. Screening has been recommended, but the benefit of population-based screening has never been shown in a randomized clinical trial. In this article, we estimate the benefit of screening young men, using a theoretical model. METHODS: A phenotypic screening scenario was modelled using a decision tree. Gain of quality-adjusted life-years was used as a measure of benefit, and estimated using Markov processes. Data on the accuracy of the screening tests, the prevalence of HH and the risk of liver cirrhosis were mainly derived from a cross-sectional study on the prevalence and morbidity of HH in 30509 men. Data on the excess mortality of cirrhosis were taken from relevant literature. Sensitivity analysis was done for important variables. RESULTS: Assuming basal case values for variables, screening a cohort of 1000 men aged 30 years for phenotypic HH would gain about 8 quality-adjusted life-years, compared to awaiting symptomatic disease. Based on actual costs of our cross-sectional study, the screening cost was US$250 per quality-adjusted life-year gained. The prevalence of phenotypic HH, the excess mortality of liver cirrhosis, the quality of life in non-cirrhotic HH patients, and the fractions of patients compliant with treatment were the most important variables in the sensitivity analysis. CONCLUSION: Incorporating screening for phenotypic HH in health survey programmes for young men may be worthwhile.

Adult↗
PubMed2004

Development and implementation of a database system to manage a large-scale mouse ENU-mutagenesis program.

A mouse ENU-mutagenesis program at RIKEN GSC has been initiated to conduct a large-scale, genome-wide, early- and late-onset phenotypic screen of mutant mice. We screened about a hundred mice every week with a comprehensive set of phenotype assays including behavioral tests based on a modified SHIRPA protocol, blood tests (both clinical biochemical testing and hemogram), and measurement of locomotor activity in their home cages. To manage the entire program, we developed a client/server architecture database system and named it MUSDB (Mutagenesis Universal Support DataBase). It manages mouse husbandry, mating protocols, procedures for ENU injection and phenotypic screens, phenotype inheritance tests, preservation of sperm and organs, and other materials generated during the program. We have implemented MUSDB in quite a large-scale system that includes 150 client computers. It has, helped reduce typographical errors and provided simple and efficient operation via its front-end user interface. It significantly contributed to the communication within and between workgroups in the program and in the accumulation of various phenotypic and inheritance data.

Animals↗
PubMed1998

A phenotype-based screen for embryonic lethal mutations in the mouse.

The genetic pathways that control development of the early mammalian embryo have remained poorly understood, in part because the systematic mutant screens that have been so successful in the identification of genes and pathways that direct embryonic development in Drosophila, Caenorhabditis elegans, and zebrafish have not been applied to mammalian embryogenesis. Here we demonstrate that chemical mutagenesis with ethylnitrosourea can be combined with the resources of mouse genomics to identify new genes that are essential for mammalian embryogenesis. A pilot screen for abnormal morphological phenotypes of midgestation embryos identified five mutant lines; the phenotypes of four of the lines are caused by recessive traits that map to single regions of the genome. Three mutant lines display defects in neural tube closure: one is caused by an allele of the open brain (opb) locus, one defines a previously unknown locus, and one has a complex genetic basis. Two mutations produce novel early phenotypes and map to regions of the genome not previously implicated in embryonic patterning.

Animals↗
PubMed2000

[Investigative application of a laboratory database].

An end user computing system (EUC) is usually part of the laboratory information system. Effective use of this EUC system is described in this article. The first application of this system is for monitoring the turn-around time (TAT) of laboratory work flow. Precise time sequential data were analyzed using this end user computing system. Weekly fluctuations of TAT depended on the cyclic changes in requisition numbers of test samples. Another application of EUC was phenotype screening of genetic abnormalities. Screenings from high triglyceride and low HDL cholesterol were effective for the phenotype screening for genetic lipoprotein lipase abnormalities, and phenotype screening from high albumin cholinesterase ratio was effective for genetic abnormalities causing silent cholinesterase. Investigational application of EUC was demonstrated in two areas of laboratory research.

Cholinesterases↗
PubMed2005

Phenotype-first screening for the identification of novel drug targets.

Modern drug discovery is predominantly a target-driven process, where success is intricately linked to the selection of an appropriate molecular target. Ideally, there is conclusive functional evidence that a selected target is disease-relevant and, furthermore, suitable for drug development. Phenotype-first screening is a highly attractive approach for target identification because it offers the unique possibility to analyse entire genomes in an unbiased fashion for disease-related phenotypes. Various studies have demonstrated that phenotype-first screening can be successfully applied to the identification of drug targets, thus establishing this approach as a valuable tool for future target discovery efforts.

Animals↗
PubMed1999

Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen.

Small molecules that perturb specific protein functions are valuable tools for dissecting complex processes in mammalian cells. A combination of two phenotype-based screens, one based on a specific posttranslational modification, the other visualizing microtubules and chromatin, was used to identify compounds that affect mitosis. One compound, here named monastrol, arrested mammalian cells in mitosis with monopolar spindles. In vitro, monastrol specifically inhibited the motility of the mitotic kinesin Eg5, a motor protein required for spindle bipolarity. All previously known small molecules that specifically affect the mitotic machinery target tubulin. Monastrol will therefore be a particularly useful tool for studying mitotic mechanisms.

Actins↗
PubMed2005

Factors affecting the uptake of screening: a randomised controlled non-inferiority trial comparing a genotypic and a phenotypic strategy for screening for haemochromatosis.

BACKGROUND/AIMS: Haemochromatosis provides an example where a novel pragmatic genotypic screening strategy may be compared with a phenotypic strategy assessing factors affecting uptake, feasibility and cost. METHODS: A randomised controlled 'non-inferiority' trial testing the hypothesis that the uptake of testing in the genotypic strategy would not be inferior to the uptake in a phenotypic screening strategy. Three thousand individuals aged 30-70 were randomly selected and randomly allocated (stratified by age and sex) to one of two screening strategies. Phenotypic-transferrin saturation on blood sample taken at GP surgery or genotypic-saliva sample taken at home; followed in screen positive individuals with assessment of iron status and genotyping. RESULTS: The difference in uptake between the two strategies was 3.4% (95% CI=0.5-6.8). Uptake was low (32%) and least in young men from socially deprived areas. Phenotypic screening was least costly. CONCLUSIONS: In this study, investigating the uptake of screening for a treatable disease in primary care, the uptake of screening with the genotypic strategy was not inferior to that in the phenotypic strategy. The poor uptake in younger men would further limit the effectiveness of screening for haemochromatosis and may have implications for other screening programmes targeted to this group.

Adult↗
PubMed2003

Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.

Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.

Cells, Cultured↗
PubMed2006

Mutagenesis as an unbiased approach to identify novel contraceptive targets.

To accommodate diverse personal needs in family planning, diverse contraceptive approaches are desirable. This goal requires identification of new contraceptive targets. Phenotype-driven mutagenesis is an unbiased approach to identify novel genes and functions in reproductive processes. The ReproGenomics Program at The Jackson Laboratory is a United States National Institutes of Health resource for production, identification and distribution of mutant mouse models of infertility that can be used for identification of potential targets for contraception. The strategy of this program is whole genome, random ENU mutagenesis, coupled with a phenotype screen for breeding failure as the only phenotype. A three-generation breeding scheme selects recessive mutations affecting reproductive functions. G3 males and females that fail to reproduce by natural mating to wild-type animals undergo secondary phenotype screens to assess gonad and accessory organ histology, hormone production, gamete production and gamete function in fertilization. The genetic transmission of the infertility trait in each family is confirmed and each mutation is genetically mapped to a defined chromosome region, facilitating identification of candidate genes from sequence and expression databases. Genes essential for fertility in both males and females and acting both meiotically and post-meiotically have been identified by this strategy. Phenotypes include male infertility with normal sperm count, but failure in fertilization of oocytes. Phenotype descriptions of each mutation are posted on the program website, . These unique reproductive mutant mouse resources will lead to new discoveries in andrology (and gynecology) research, as well as reproductive medicine. Dissection of gene function in known and newly discovered reproductive pathways will expand our focus to reveal novel targets for contraception.

Alkylating Agents↗
PubMed2005

Screens using RNAi and cDNA expression as surrogates for genetics in mammalian tissue culture cells.

We have developed methods for the automation of transfection-grade DNA preparation, high-throughput retroviral preparation, and highly parallel phenotypic screens to establish approaches that will allow investigators to examine in an unbiased manner the roles of proteins in mammalian cells. These methods have been used to raise or lower the levels of individual kinases in individual micro-well cultures either by cDNA or short hairpin RNA expression and will allow investigators to treat mammalian cells in culture in manners that are analogous to genetic screens in yeast. Our proof-of-principle experiments have been performed in human cells using repositories that represent over 75% of the protein, nucleotide, carbohydrate, lipid, and amino acid kinases in the human genome. These initial experiments have demonstrated the feasibility of two general types of screens. We have performed phenotypic screens to identify proteins with specific roles in a chosen function and genetic interaction screens to establish epistatic relations between different proteins. The results suggest that any phenotype that can be scored by a robust assay in tissue culture is amenable to these types of screens and that interactions between mammalian proteins can be established. These results point to the near-term goal of establishing comprehensive, unbiased screens that will allow queries on the roles of all human proteins.

Carcinoma, Non-Small-Cell Lung↗
PubMed2005

A battery of olfactory-based screens for phenotyping the social and sexual behaviors of mice.

We provide a battery of screens that reflect the social and sexual behavior displayed by both captive and feral mice. Some screens focus on measuring aspects of chemical communication, providing information about whether or not mice will interact and if they do so, predict the nature of the interaction. Other screens measure direct interactions between target mice and same- and opposite-sex conspecifics, providing an indication of the social status and sexual responsiveness, respectively, of target mice. The battery of screens yields a high-throughput bioassay of a mouse's relative social status, competitive ability, social discrimination, and sexuality. These traits are essential elements of the socio-sexual behavior of mice as well as humans. Thus, by identifying phenotypic deviants for complex behaviors we will allow geneticists to map behavioral abnormalities onto specific chromosomes and increase the efficacy of genetically altered mice as models for human behavioral disorders.

Agonistic Behavior↗
PubMed2007

Power matters in closing the phenotyping gap.

Much of our understanding of physiology and metabolism is derived from investigating mouse mutants and transgenic mice, and open-access platforms for standardized mouse phenotyping such as the German Mouse Clinic (GMC) are currently viewed as one powerful tool for identifying novel gene-function relationships. Phenotyping or phenotypic screening involves the comparison of wild-type control mice with their mutant or transgenic littermates. In our study, we explored the extent to which standardized phenotyping will succeed in detecting biologically relevant phenotypic differences in mice generated and provided by different collaborators. We analyzed quantitative metabolic data (body mass, energy intake, and energy metabolized) collected at the GMC under the current workflow, and used them for statistical power considerations. Our results demonstrate that there is substantial variability in these parameters among lines of wild-type C57BL/6 (B6) mice from different sources. Given this variable background noise in mice that serve as controls, subtle phenotypes in mutant or transgenic littermates may be overlooked. Furthermore, a phenotype observed in one cohort of a mutant line may not be reproducible (to the same extent) in mice coming from a different environment or supplier. In the light of these constraints, we encourage researchers to incorporate information on intrastrain variability into future study planning, or to perform advanced hierarchical analyses. Both will ultimately improve the detectability of novel phenotypes by phenotypic screening.

Animals↗
PubMed2006

Phenotype-based screening of mechanistically annotated compounds in combination with gene expression and pathway analysis identifies candidate drug targets in a human squamous carcinoma cell model.

The squamous cell carcinoma HeLa cell line and an epithelial cell line hTERT-RPE with a nonmalignant phenotype were interrogated for HeLa cell selectivity in response to 1267 annotated compounds representing 56 pharmacological classes. Selective cytotoxic activity was observed for 14 of these compounds dominated by cyclic adenosine monophosphate (cAMP) selective phosphodiesterase (PDE) inhibitors, which tended to span a representation of the chemical descriptor space of the library. The PDE inhibitors induced delayed cell death with features compatible with classical apoptosis. The PDE inhibitors were largely inactive when tested against a cell line panel consisting of hematological and nonsquamous epithelial phenotypes. In a genome-wide DNA microarray analysis, PDE3A and PDE2A were found to be significantly increased in HeLa cells compared to the other cell lines. The pathway analysis software PathwayAssist was subsequently used to extract a list of proteins and small molecules retrieved from Medline abstracts associated with the hit compounds. The resulting list consisted of major parts of the cAMP-protein kinase A pathway linking to ERK, P38, and AKT. This molecular network may provide a basis for further exploitation of novel candidate targets for the treatment of squamous cell carcinoma.

Carcinoma, Squamous Cell↗
PubMed2004

MuTrack: a genome analysis system for large-scale mutagenesis in the mouse.

BACKGROUND: Modern biological research makes possible the comprehensive study and development of heritable mutations in the mouse model at high-throughput. Using techniques spanning genetics, molecular biology, histology, and behavioral science, researchers may examine, with varying degrees of granularity, numerous phenotypic aspects of mutant mouse strains directly pertinent to human disease states. Success of these and other genome-wide endeavors relies on a well-structured bioinformatics core that brings together investigators from widely dispersed institutions and enables them to seamlessly integrate data, observations and discussions. DESCRIPTION: MuTrack was developed as the bioinformatics core for a large mouse phenotype screening effort. It is a comprehensive collection of on-line computational tools and tracks thousands of mutagenized mice from birth through senescence and death. It identifies the physical location of mice during an intensive phenotype screening process at several locations throughout the state of Tennessee and collects raw and processed experimental data from each domain. MuTrack's statistical package allows researchers to access a real-time analysis of mouse pedigrees for aberrant behavior, and subsequent recirculation and retesting. The end result is the classification of potential and actual heritable mutant mouse strains that become immediately available to outside researchers who have expressed interest in the mutant phenotype. CONCLUSION: MuTrack demonstrates the effectiveness of using bioinformatics techniques in data collection, integration and analysis to identify unique result sets that are beyond the capacity of a solitary laboratory. By employing the research expertise of investigators at several institutions for a broad-ranging study, the TMGC has amplified the effectiveness of any one consortium member. The bioinformatics strategy presented here lends future collaborative efforts a template for a comprehensive approach to large-scale analysis.

Animals↗