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High throughput primary phenotyping (WP1)

Aims and Overview

We propose to enter a large number of mutant lines into a new broad-based primary phenotyping screen - EMPReSSslim. EMPReSSslim has been developed from the collection of SOPs generated and validated under the EUMORPHIA programme. EMPReSSslim is designed to cover most body systems and to provide a comprehensive first-pass investigation of mouse mutant phenotype that will form the basis for more in-depth phenotyping by members of the EUMODIC consortium, or by other interested labs within the wider genetics community. An early but important part of the programme will carry out phenotype comparisons for a selection of mutations on two genetic backgrounds - 129 and BL/6. Data from primary phenotyping will be entered into the EuroPhenome database.

Design of EMPReSSslim

EMPReSS, a robust set of validated phenotyping SOPs, produced as part of the EUMORPHIA programme has been refined and slimmed in order to develop a new protocol, EMPReSSslim, for primary phenotype screens (see Fig. 4). EMPReSSslim comprises two integrated pipelines of tests, each commencing at 8 weeks and terminating at 14 weeks. The tests encompass dysmorphology; blood and metabolic phenotypes; bone disease; immunology and allergy; cardiovascular; neurology and behaviour; and sensory and neuromuscular systems. The precise order and timing of these screens is still under discussion by the consortium. The test series are carefully designed to eliminate interference between the various phenotyping modalities applied. So, for example, in Pipeline 1 sufficient time periods have been built into the scheme between tests requiring blood collection to comply with necessary health and welfare standards, as well as preventing residual effects of protocols on subsequent tests. Pipeline 2 from 8-10 weeks covers a series of neurological, behavioural and sensory tests. The order and timing for these screens has been standardised and validated across 5 European centres as part of the EUMORPHIA programme. However, the final composition and order of tests in EMPReSSslim is being review by an expert panel drawn from EUMODIC partners, to determine if the number of tests in this set of screens can be reduced and still reliable identify neurobehavioural and sensory mutants.

EMPReSSslim

Most of the proposed tests in EMPReSSslim have been designed and validated under the EUMORPHIA programme. We do not include a lengthy explanation of every test: the SOP, available on the EMPReSS website (www.eumorphia.org), includes details of test purpose and procedures. For convenience we include here the SOP identifier.

Pipeline 1 comprises tests for:

9 weeks - Dysmorphology: see EMPReSS SOP 12_003. Dysmorphology screens are simple to undertake by providing important information on a variety of craniofacial, limb, and other visible malformations as well as visible phenotypes such as coat colour/texture, genital irregularities and dentition.

10 weeks - Indirect calorimetry: this is a new test not previously undertaken by EUMORPHIA for which a new SOP is being developed and validated. Open-circuit indirect calorimetry provides measures of O2 consumption, CO2 production, the respiratory quotient and heat. It gives the most accurate measurement of energy generated and calories burned by the body, and is essential in characterizing energy homeostasis. Importantly, the current fully automated commercial systems are relatively hands-free and high throughput.

11 weeks - Non-invasive blood pressure: see EMPReSS SOP 5_001. Using the tail cuff method, we will make an assessment of systolic blood pressure and heart rate in conscious mice.

12 weeks - Echocardiography: see EMPReSS SOP 5_006. This test will allow us to assess cardiac anatomy and function (systolic and diastolic) in anaesthetised mice using echocardiography. The inclusion of this test is still under discussion.

13 weeks - simplified IPGTT: see EMPReSS SOP 4_003. A simplified, high throughput variant of the Intra-Peritoneal Glucose Tolerance Test will be employed.

13 or 14 weeks - DEXA: see EMPReSS SOP 12_004. DEXA scanning will be employed as a terminal procedure to assess bone mineral content and density and fat content.

13 or 14 weeks - X-ray: see EMPReSS SOP 12_006. Following DEXA scanning the mouse carcass will be X-rayed to record skeletal structure using a FAXITRON X-ray system.

Pipeline 2 comprises tests for:

9-12 weeks - Integrated series of tests exploring neurological, sensory and behavioural function: see EMPReSS SOPs 10_001 - 10_009. As alluded to above, EUMORPHIA has designed an integrated battery of tests exploring these systems that has been designed to prevent interference with the outcome of subsequent tests. The exact composition and sequence of these tests will be determined by a EUMODIC working group using this information. It is likely that they will be Open Field (10_001), Modified SHRIPA (10_002), Grip Strength (10_003), Rotarod (10_009), Acousitc Startle and PPI (10_005), Hot Plate (SOP under development).

13 weeks - Ophthalmoscope and slit lamp: see EMPReSS SOPs 8_003 and 8_004. These two protocols assess both abnormalities of the fundus (such as retinal degeneration) as well as anterior segment defects affecting the iris, cornea and lens.

13 or 14 weeks - terminal blood collection for: Clinical chemistry: see EMPReSS SOP 3_007. Routine clinical chemistry screens employing the Olympus AU400 autoanalyser provide substantive information on a variety of clinical chemical blood parameters, specific substrates and electrolytes.

Haematology: see EMPReSS SOP 3_008. Depending upon local licensing rules that govern the maximum volume of blood taken in any one monthly period, a haematological analysis will be undertaken either at 12 weeks, or shortly preceding simplified IPGTT at 14 weeks. Determination of blood cell counts (white blood cells, red blood cells, platelets), haemaglobin measurement and the calculation of haematological indices will be carried out.

FACs analysis of peripheral blood cells and Ig concentrations: see EMPReSS SOPs 6_004 and 6_005. Both these tests will provide a preliminary exploration of immunological function and allergy.

ANP levels: Levels of ANP in blood are an excellent indicator of heart muscle dysfunction. EUMODIC will develop an SOP for measuring ANP levels in blood. The test will be included in EMPReSSslim once it has been validated.