The International Mouse Phenotyping Consortium (IMPC) web portal (http://www.mousephenotype.org) provides the biomedical community with a unified point of access to mutant mice and rich collection of related emerging and existing mouse phenotype data. IMPC mouse clinics worldwide follow rigorous highly structured and standardized protocols for the experimentation, collection and dissemination of data. Dedicated ‘data wranglers’ work with each phenotyping center to collate data and perform quality control of data. An automated statistical analysis pipeline has been developed to identify knockout strains with a significant change in the phenotype parameters. Annotation with biomedical ontologies allows biologists and clinicians to easily find mouse strains with phenotypic traits relevant to their research. Data integration with other resources will provide insights into mammalian gene function and human disease. As phenotype data become available for every gene in the mouse, the IMPC web portal will become an invaluable tool for researchers studying the genetic contributions of genes to human diseases.

Although next generation sequencing has revolutionised the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by our lack of knowledge of function and pathobiological mechanism for most genes. To address this challenge, the International Mouse Phenotyping Consortium (IMPC) is creating a genome- and phenome-wide catalogue of gene function by characterizing new knockout mouse strains across diverse biological systems through a broad set of standardised phenotyping tests, with all mice made readily available to the biomedical community. Analysing the first 3328 genes reveals models for 360 diseases including the first for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations are novel, providing the first functional evidence for 1092 genes and candidates in unsolved diseases such as Arrhythmogenic Right Ventricular Dysplasia 3. Finally, we describe our role in variant functional validation with the 100,000 Genomes and other projects.

Pulsed-field gel electrophoresis (PFGE) has become the gold standard of molecular methods in epidemiological investigations. In spite of its high resolving power, use of the method has been hampered by inadequate laboratory-to-laboratory reproducibility. In the project described here we have addressed this problem by organizing a multilaboratory effort in which the same bacterial strains (subtype variants of the Iberian and Brazilian methicillin-resistant Staphylococcus aureus--MRSA--clones) were analyzed by twenty investigators in thirteen different laboratories according to an indentical protocol, which is reproduced here in detail. PFGE patterns obtained were analyzed at a central laboratory in order to identify specific technical problems that produced substandard macrorestriction patterns. The results including the specific technical problems and their most likely causes are described in this communication. Also listed are seven major epidemic clones of MRSA which have been characterized by molecular fingerprinting techniques and the prototypes of which have been deposited at the American Type Culture Collection, from where they will be available for interested investigators for the purpose of typing MRSA isolates. It is hoped that this communication will contribute to the improvement of the reproducibility and technical/aesthetic quality of PFGE analysis.

All clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates examined so far contain the mecA gene, a 2130bp stretch of DNA of non-staphylococcal origin which, together with a larger block (up to 40-60 Kb) of 'foreign' DNA, is incorporated into the staphylococcal chromosome. mecA encodes for the 78 Kd penicillin-binding protein (PBP) 2A, which has very low affinity for beta-lactam antibiotics. The sequence of the mecA gene contains structural motifs characteristic of cell wall synthetic transpeptidases. It is generally assumed that the mecA gene product (PBP 2A) acts as a surrogate enzyme which takes over the task of cell wall synthesis from the normal complement of staphylococcal PBPs, since the latter are inhibited by relatively low (e.g. methicillin) concentrations of beta-lactam antibiotics. While direct biochemical evidence for a transpeptidase activity in PBP 2A is still missing, the essentiality of an intact mecA gene for the expression of high-level methicillin resistance has been clearly established by transposon inactivation experiments. On the other hand, it was already noted some time ago that an intact mecA and its gene product PBP 2A alone cannot be fully in control of the resistant phenotype, since all MRSA isolates, irrespective of their MIC values (from as low as 3 mg/L or as high as 1600 mg/L), were found to contain comparable amounts of PBP 2A. Such major disparities between cellular amounts of PBP 2A and the antibiotic MIC values suggested that a factor or factors of unknown nature ('factor X') other than the mecA gene product also played an essential role in the phenotypic expression of resistance. The same conclusion was reached in early genetic studies in which methicillin resistance could be reduced by insertional inactivation of a chromosomal site (omega 2003) within the so-called femA gene--(factor essential for the expression of methicillin resistance) outside the mecA determinant. More recently, several additional chromosomal sites were identified outside the mecA gene in which transposon inactivation reduced the level of beta-lactam resistance. The importance of these genes becomes clear if one realizes that it is the appropriate functioning of these determinants (in the genetic background of MRSA) rather than the quantity of PBP 2A in the cells that seems to determine the MIC value of an MRSA isolate. It is not clear at the present time how many such 'auxiliary genes' exist and exactly how these gene co-operate with the mecA gene in bringing about high-level beta-lactam resistance.(ABSTRACT TRUNCATED AT 400 WORDS)

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The Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines were developed to address the lack of reproducibility in biomedical animal studies and improve the communication of research findings. While intended to guide the preparation of peer-reviewed manuscripts, the principles of transparent reporting are also fundamental for in vivo databases. Here, we describe the benefits and challenges of applying the guidelines for the International Mouse Phenotyping Consortium (IMPC), whose goal is to produce and phenotype 20,000 knockout mouse strains in a reproducible manner across ten research centres. In addition to ensuring the transparency and reproducibility of the IMPC, the solutions to the challenges of applying the ARRIVE guidelines in the context of IMPC will provide a resource to help guide similar initiatives in the future.

Competitive hybridization was used to detect the deletion of chromosomal DNA accompanying the loss of resistance to methicillin (and concomitantly, to cadmium, mercury and tetracycline) from a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA). The method was also used to screen a partial plasmid library of chromosomal HindIII fragments from the MRSA strain. Eight recombinant plasmid clones were identified as containing DNA included in the deletion. These clones were used as probes to screen a phage library of the total DNA of the same MRSA strain, resulting in the isolation of overlapping recombinant phage clones carrying 24 kb of the deleted DNA. Two of the cloned HindIII fragments were associated closely with methicillin resistance, as shown by probing DNA from an independent methicillin-sensitive/resistant transduced strain pair and from two MRSA strains following growth in the presence of high concentrations of methicillin. The endonuclease map of the cloned DNA indicates the presence of four copies of a direct repeat less than 1 kb in size. The map is also consistent with the presence in the chromosome of sequences for mercury resistance (mer A mer B) and for tetracycline-resistance plasmid pT181.

Five cDNA clones were isoliited from barley {Hordeum vulgare L.) that encoded niRNAs related to xyloglucan endotransglycosylase (XET). One of the clones encoded a protein with XET activity in vitro. Sequence comparisons revealed five families of XET-related sequences, one of which (containing two of the barley genes) was novel. Hybridization studies using clone-specific probes indicated tbat the corresponding genes were represented once, or possibly twice, in the barley genome. Treatment of dwarf mutants with gibberellic acid (GA,), or boniozygosity at tbe 'slender' {sliiL) locus, resulted in a 2'5-fold (approximately) stimulation of blade elongation rate. Tbree of the five clones detected niRNAs that were maximally expressed towards the base of the blade, and present in greater quantities in GAj-treated or slender seedlings. The remaining two clones detected niRNAs tbat were maximally expre.ssed in the middle of the blade. Relative elemental growth rate (REGR) profiles of leaves growing with or without GA, treatment revealed similar maximal REGR values despite a 2-5-fold difference in leaf elongation rate. Segments of GA3-treated leaves attained tbeir nuiximal REGR values more rapidly, this being associated with enhanced expression of tbe three 'basal' XET-related mRNAs. Highest XET activities were detected in tbe base of tbe elongation zone, and in GA3-treated seedlings a ,second activity peak was observed near tbe di.stal end of the elongation zone. We conclude that there are likely to be several XET isoenzymes witb different expression patterns, and identify those XET-related proteins potentially involved in leaf elongation.