Man or mouse

When it comes to understanding and modeling human disease, clinical chemistry of the mouse has proved crucial. Here Tertius Hough tells us how this humble rodent can unlock the functional genomics of disease.  

IN the post-genomic era the focus of mammalian genetic research has shifted to the study of gene function and expression and those genes relevant for the diagnosis, prevention and therapy of human diseases are of particular interest. The mouse has become the organism of choice for mammalian genetic studies so continues to play a pivotal role in this quest. It has been the most widely used organism to model human disease and is especially suitable for this because genomic DNA organisation and gene expression patterns have been highly conserved between the species. Regions of conserved synteny have also been well documented and are useful in identifying homologous single gene disorders between mouse and man. This is because products of orthologous genes are generally involved in the same physiological and cell biological pathways. In addition, all mammals have similar physiological and metabolic systems.  Our detailed understanding of mammalian biochemical pathways and the underlying changes associated with various disease conditions make biochemical screens a very powerful tool in phenotypic analysis of mouse models of human disease. Knowledge of biochemical abnormalities associated with certain human disease conditions provides markers for the identification of mice that may be useful in modeling similar inherited disorders.

The study of gene function relies heavily on the genetic dissection of biological processes in experimental animals. Spontaneous and induced mutations have been studied in the mouse for more than a century but in recent years there has been an explosion in their use. By studying mutations in a gene we can gain insight into the functions of that gene in biological systems.  The induction of point mutations include the possibilities of uncovering partial gain and loss of functional alleles unlike gene knockout models that only mimic null phenotypes. One of the main approaches is the use of genome-wide chemical mutagenesis. The Harwell Mutagenesis Program conducted in the MLC generates large numbers of mice subject to comprehensive biological and clinical phenotyping screens. These screens are designed to identify specific abnormalities associated with various classes of human disease conditions of interest to both the research groups within the MGU and the various external / collaborative programmes. The variety of tests offered by the clinical laboratory are incorporated into many of these high throughput screens.

An Olympus AU400 Clinical Chemistry analyser is used at Harwell to perform standard biochemical profiles

In recent decades advances in diagnostic techniques and the incorporation of automated screening methods that require as little as 2 ?l of sample for biochemical testing, have made extensive clinical chemical testing of mouse blood samples practically feasible. At Harwell an Olympus AU400 automated clinical chemistry analyser equipped with an ion selective electrode is used to perform standard biochemical profiles on plasma, serum and urine samples. The semi-automated AU400 is a common sight around NHS clinical laboratories but is also suited for the analysis of mouse samples following minor adjustments to the probes and adaptation of the sample cups. Paediatric parameter settings are available with most of the reagent kits which helps to economise on the required sample volume.

Because of their small size, the collection of an adequate mouse blood sample of acceptable quality can be challenging – especially if an extensive profile of tests are desired. A Thermacage in which the mice are exposed to circulating warm air is often used to encourage vasodilation prior to bleeding which facilitates sampling. Blood collection in the mouse is a regulated procedure and therefore strictly controlled and monitored by the Home Office. At Harwell, samples from live mice are collected from a superficial incision in the lateral tail vein under local anaesthesia (up to 300μl). Alternative post mortem methods include cardiac punctures (although such samples are very often haemolysed) and blood collection from the jugular vein.  Paediatric collection tubes are ideally suited for the small volumes collected. Results for clinical chemistry tests can vary greatly between the various inbred strains of mice used in functional genetic studies. Various other factors including age, sex, diet and the time of sample collection can also induce marked variation. Since the literature contains limited information on normal ranges it is often desirable to produce suitable control data for each specific study. In fact, when phenotyping established mutant lines unaffected (wild type) littermates are often best suited as controls.  High-throughput programmes however often rely on a running mean +/- 2-3 standard deviations as a cut-off point to identify significant outliers.

The clinical chemistry laboratory provides a phenotyping service to the various groups that constitute the MGU as well as offering a service to research groups on site and in the wider scientific community. The MLC currently hosts several high-throughput screening programmes with a major clinical chemistry component, including large phenotype screens for skeletal, liver and renal disorders that utilise the chemical mutagenesis pipelines. The laboratory has an established track record dating back to the first Harwell mutagenesis programme. The original clinical chemistry screen processed ~2000 plasma samples from the first generation offspring of mutagenised mice. The selected 17 test profile included liver function tests (ALT, AST, Total Protein and Albumin), bone profile (Calcium, Inorganic Phosphorus and Alkaline Phosphatase), kidney profile (Urea, Creatinine, Sodium, Potassium and Chloride) as well as a lipid profile (Total Cholesterol, HDL Cholesterol and Triglycerides) and glucose. The latter tests proved especially fruitful as several lipidaemic and diabetic mice were identified by the screen. The relatively high frequency of abnormal results for cholesterol and glucose reflects the polygenic involvement in the homeostasis of lipids and glucose. Mice that displayed consistently abnormal results were selected for inheritance testing. The rate of observation of abnormal phenotypes for this screen was high (1 in 33 mice screened) and was possibly a reflection of the multiple tests included on the panel. Nevertheless, it is clear that the high number of uncovered phenotypes validate the use of clinical chemistry screens to identify mice with metabolic abnormalities. For lines where inheritance testing has been completed, we found that over half of the abnormal blood phenotypes were inherited. The screen also identified models of rare diseases including Tangier’s disease and adult hypophosphatasia.

In 2004 the Medical Research Council opened its new state of the art mouse house facility, the Mary Lyon Centre (MLC) at Harwell. The £18m centre, which can house up to 65,000 mice, was purpose built to act as a central platform for the development of functional genomics in the UK.  The MLC combines expertise in mouse production and archiving, mutagenesis, transgenesis and phenotyping in a high health status environment thereby providing the ideal tools and space for the development of mouse models of human disease (www.mlc.har.mrc.ac.uk). The centre serves the needs of the Harwell Mammalian Genetics Unit (www.mgu.har.mrc.ac.uk) and the wider UK and international scientific community. The clinical laboratory in the Pathology Department specialises in mouse clinical chemistry and provides one of several integrated phenotyping platforms employed to identify and characterise mouse models in the MLC.

The use of metabolic cages in which mice are singly housed allow the collection of urine over a 24 hour period which facilitates the identification and characterisation of mice with renal abnormalities. The cages are designed to separate urine and fecal matter which drops through the mesh base on which the animals are kept. The standard profile of urine tests performed on the AU400 includes sodium, potassium, chloride, urea, creatinine, calcium, inorganic phosphate, urinary CSF protein and glucose.  Dilutions are often required to bring certain parameters into range. This is frequently the case with urine potassium. In conjunction with plasma samples collected from the same sets of mice, urine samples from metabolic cages provide essential phenotypic information for the screens for renal disorders in the MLC.

The clinical laboratory recently acquired a CANTO II FACS analyser from BD Biosciences. This cytometer has 6 lasers and can distinguish 8 colours per well. It is equipped with a high throughput sampler which allows samples to be loaded in 96 well plate format.  FACS analysis is frequently used to monitor populations of peripheral blood cells in immunological mutant lines.  The data can be complemented by ELISA assays that measure the acquired and innate immune responses of mutant vs control cohorts following immune challenges. Another recent powerful and versatile addition to the laboratory is a Bioplex multiplex analysis system (Biorad) that permits the simultaneous analysis of many different biomolecules (proteins, peptides, or nucleic acids) in a single microplate well. Applications include Molecular biology, immunoassays, receptor-ligand and enzymatic assays.  In the MLC the system is currently used for endocrine, cytokine and immunoglobulin profiles, although this is sure to expand due to the appeal of the system’s low sample volume requirements. A variety of beads with specific assay combinations are commercially available.

Various European-wide and international efforts are now being made to expand the existing mouse mutant resource that will accelerate insights into human disease. The MGU initiated several such EU-funded programmes and the MLC has close involvement with a few of these.  The Eumorphia Programme (European Union Mouse Research for Public Health and Industrial Applications) which ran from 2002 to 2006 led to the development of a database of Standard Operating Procedures (SOPs) for screens that can be used to phenotype mice (www.eumorphia.org). These SOP’s are freely available (see www.empress.har.mrc.ac.uk) and referred to as the European Mouse Phenotyping Resource of Standardised Screens (EMPReSS). These SOP’s have already led to improved characterisation of mouse models for the understanding of human disease and allow for better comparison of results between various research institutions across Europe. The European Mouse Disease Clinic (Eumodic) is a new €12m European Commission funded project. The 4 year programme will generate phenotypic data on 650 knockout mouse lines using selected EMPReSS SOP’s The consortium consists of 18 participating laboratories in 8 countries and is coordinated by the MRC Harwell (www.eumodic.eu). 

The MLC’s involvement in Eumodic has facilitated the influx of new technologies into the clinical laboratory. The MGU and MLC will aim to phenotype roughly 50 knockout mouse lines each year. Screening will include morphological, metabolic, cardiovascular, skeletal, neurobehavioural and sensory platforms. The clinical laboratory will be involved in the clinical chemistry, haematology and immune platforms. The standard tests will include a clinical chemistry panel of roughly 20 tests, a comprehensive isotyping panel to be analysed using Bioplex bead array, an ELISA assay for atrial natriuric peptide (ANP) as part of the cardiac screen, detailed examination of peripheral blood cells using the CANTO II and finally haematology.

In collaboration with external groups the clinical laboratory is now expanding into the field of haematology and recently acquired a Siemens Advia 2120 for full blood counts and differentials.  The Advia 2120 is capable of measuring over 30 parameters from a 175µl whole blood sample.  The multispecies software provided with the analyser includes settings for the analysis of certain inbred mouse strains. Over the past year the laboratory has also established collaborative links in the fast growing field of metabolomics. Aliquots of plasma and urine samples from the first generation mutagenesis pipeline as well as samples from established mouse models have already been sent to the Department of Biochemistry at the University of Cambridge where high resolution 1H Nuclear Magnetic Resonance (NMR) spectroscopy and gas chromatography mass spectroscopy (GCMS) is used to generate more detailed metabolic fingerprints from plasma and urine samples (www.bioc.cam.ac.uk/uto/griffin). These techniques promise to reveal new classes of biochemical phenotypes thereby maximising the use and potential of the mice bred in the MLC.

A vast selection of mutant mice, the majority of which were identified on various mutagenesis programmes, are already at the disposal of the scientific community (see for example www.mgu.har.mrc.ac.uk, www.jax.org, www.gsf.de, www.gsc.riken.go.jp). The European Mouse Mutant Archive (EMMA) aims to collect, archive and distribute European mouse mutant strains that are essential for basic biomedical research (www.emma.rm.cnr.it). These growing mouse mutant archives are a valuable resource for the identification of novel genes underlying various human diseases. The increasing number of commercially available mouse-specific kits for the measurement of biomarkers of disease reflects the escalating emphasis on the mouse as model organism in biomedical research. As advances in technology allow the researcher to obtain more detailed biological information from smaller samples and more test options with improved specificity become available, clinical chemistry appears set to play an important role in functional genomics.

By, Tertius Hough manages the Mary Lyon Centre’s clinical laboratory at the MRC Harwell in Oxfordshire.