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1.  A systematic genome-wide analysis of zebrafish protein-coding gene function 
Nature  2013;496(7446):494-497.
Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at an enormous rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in vertebrate model organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches1-3 and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes3,4, this number falls significantly short of all >22,000 mouse protein-coding genes5. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning6, insertional mutagenesis7-9, antisense morpholino oligonucleotides10, targeted re-sequencing11-13 and zinc finger and TAL endonucleases14-17 have made significant contributions to our understanding of the biological activity of vertebrate genes, but the number of genes studied again falls well short of the >26,000 zebrafish protein-coding genes18. Importantly, for both mice and zebrafish, none of these strategies is particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Enabled by a well-annotated zebrafish reference genome sequence18,19, high-throughput sequencing and efficient chemical mutagenesis, we describe an active project that aims to identify and phenotype disruptive mutations in every zebrafish protein-coding gene. Thus far we have identified potentially disruptive mutations in more than 38% of all known protein coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.
PMCID: PMC3743023  PMID: 23594742
2.  Use of Purified Clostridium difficile Spores To Facilitate Evaluation of Health Care Disinfection Regimens▿ †  
Applied and Environmental Microbiology  2010;76(20):6895-6900.
Clostridium difficile is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of C. difficile that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious C. difficile spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified C. difficile spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure spores and blocking spore-mediated transmission. Complete inactivation of 106 pure C. difficile spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with C. difficile spores (17 to 29 spores/cm2) and block host transmission. Thus, pure C. difficile spores facilitate practical methods for evaluating the efficacy of C. difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control.
PMCID: PMC2953018  PMID: 20802075
3.  α-Galactosidase A-Tat Fusion Enhances Storage Reduction in Hearts and Kidneys of Fabry Mice 
Molecular Medicine  2010;16(5-6):216-221.
The protein transduction domain from human immunodeficiency virus (HIV) Tat allows proteins to penetrate the cell membrane. Enhanced cellular uptake of therapeutic proteins could benefit a number of disorders. This is especially true for lysosomal storage disorders (LSDs) where enzyme replacement therapy (ERT) and gene therapy have been developed. We developed a novel recombinant lentiviral vector (LV) that engineers expression of α-galactosidase A (α-gal A)-Tat fusion protein for correction of Fabry disease, the second-most prevalent LSD with manifestations in the brain, kidney and heart. In vitro experiments confirmed mannose-6-phosphate independent uptake of the fusion factor. Next, concentrated therapeutic LV was injected into neonatal Fabry mice. Analysis of tissues at 26 wks demonstrated similar α-gal A enzyme activities but enhanced globotriaosylceramide (Gb3) reduction in hearts and kidneys compared with the α-gal A LV control. This strategy might advance not only gene therapy for Fabry disease and other LSDs, but also ERT, especially for cardiac Fabry disease.
PMCID: PMC2864812  PMID: 20454522

Results 1-3 (3)