IMPC Phenotyping Work Group

The Phenotyping Work group consists of 5 to 6 experts from each participating member of the IMPC, along with additional external experts reflecting phenotyping expertise from the wider mouse, clinical, biotech and pharmaceutical communities. The Phenotyping Workgroup will advise the IMPC Steering Committee on the core phenotyping platforms that should be implemented as part of an IMPC primary phenotyping pipeline, taking account of the strategic needs for mouse models and the requirements of the wider biomedical, clinical, biotech and pharmaceutical communities. The work group will consider the development and inclusion of new phenotyping platforms, the timing of tests, test orders and cohort size. In addition they will advise on the statistical issues associated with the use of control cohorts, the identification of phenotypes and provide guidance on the likely costs of a core phenotyping pipeline.

Phenotype Work Group Report

A report from the IMPC Phenotyping workgroup is now available to download, and we welcome your comments and input. The report concludes by presenting a draft IMPC phenotype pipeline. However, note that the draft pipeline is at this stage an amalgam of the key primary tests that following the discussions of the Phenotype Workgroup merited serious consideration for incorporation into a final IMPC pipeline. The draft presented in Fig. 1 and 2 is a starting point for further discussion and design of a final agreed pipeline. The establishment of the final IMPC pipeline will require additional review and iterations, particularly to take account of test density, inter-test perturbations and the minimisation of these effects.

Adult Pipeline information can be found in IMPReSS.

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Comments

Andrew Blake's picture

Pipeline Organisation and Capacity:

Open Field

Ideally this needs to be done on naive animals- week 9 could be moved to week 8 and the Immunization with Ova and early blood sampling done in week 9?

This would still allow 5 weeks between the sensitization and the OVA challenge and the blood collection would be from larger animals.

Calorimetry, Plethysmography and ABR (+ fear conditioning)

To ease capacity problems- it may be valid just to perform these tests on a single sex cohort.

Cognitive test

The pipeline does not contain any cognitive tests at all. Fear conditioning (males?) could be performed on the opposite sex as the plethysmograph (females?) on week 13.

Specific of Pipeline Assays

Immune challenge

The sensitization and immune challenge by OVA and terminal haematology is considered a very important inclusion as s so much human disease has an immunological component.

ECHO and ECG

It may be an option to perform ECHO and ECG under gaseous anaesthesia for easy restraint.

Plethysmograph

If we only undertake a methacholine dose response we will not obtain any information about the lower airways and will therefore miss identifying conditions like fibrosis and COPD. One option is to measure immune cell populations on lung tissue terminally after the challenge with OVA. Another option is to leave out a methacholine dose response and just do a single CO2 exposure and plethysmography (which would give us information on lower airways). The methacoline dose response could potentially be undertaken as a secondary screen.

It is very important that we measure IgE levels as part of IMPC, both the immunology and respiratory communities will certainly want this data.

ABR

As this will time consuming, it may be important to shorten some of the existing protocols to just including a high (32kHZ) and a low (8kHz) stimulus.

IPGTT

We would like the fasted sample in the 14 week IPGTT to include insulin, this will also be replicated in the terminal blood test that should also be fasted (4-6h). I think replication is useful given the variability of the insulin assay. Insulin/Glucose measurements give an indication of insulin sensitivity.

Body Composition

Body composition would ideally be paired with the calorimetry (12 weeks) and if using EchoMRI does not require an anaesthetic. DEXA could be combined with the X-Ray (14 weeks) for body composition and does require an anaesthetic. DEXA is essential, as it will give information on bone parameters BMC/BMD.

Controls

Using littermate controls from heterozygous intercrosses has the advantage of reducing variability in the data, simpler statistical analysis etc. However this impacts on caging etc.

Pilots

The affects of different pipeline combinations would of course need to be piloted and compared to naive animals to see what the accumulatively effect of these assays is having and whether that has biological relevance.

Andrew Blake's picture

The IMPC steering committee proposes that phenotyping will be carried out a single pipeline based on 7 male +7 female cohorts. As Fig Note was described, I wonder the mice will bear with the so many phenotyping,especially too high-density schedule in 14w until 16w. According to the single pipeline, the same mice have to be subjected to immunological test,neurological test, metabolic test and so many tests. For example, since the open-field test examine an emotionality of mouse, the result of the open-field test will be affected by prior experiment, I think we had better use naive mice in this test. So, I think generally that two or three pipelines per a line are required to decrease the damage to mice from test. We will propose three pipelines including Pipeline A (Neurological-hematological), Pipeline B (morphological-clinical chemistry-Pathology), and Pipeline C (Immunology) outlined @ http://www.mousephenotype.org/IMPC_Phenotyping_comment_from_JMC2.ppt .In this case,larger number of mice should be produced.
The comments on each test are as follows.
Micronuclei Assay(6-8w)
Peripheral RBC micronuclei assay has been used as a test for detecting chronic genotoxicity of drug administration that is converted to apply in the phenotyping pipeline to search for the potential genomic instability caused by specific gene deletion. However, the feasibility of this application is rather unclear.
Dysmorphology and modified SHIRPA(9w)
Dysmorphological observations should be integrated with handling of modified SHIRPA screening. Actually, we have incorporated these two and it takes less than 15 min per a mouse.
Awake ECG / Echo (11w)
For echocardiography, clinical echo-machine still undergoes mouse measurement. It is to be discussed whether small animal-specialized machine such as Vevo770 should be introduced for more measurable parameters. To eliminate the artifacts due to anesthesia, a wake conditions is recommended, whereas animal conditions easy to handle can be attained by its administration. Otherwise, weak anesthesia or mild tranquilizer administration may be good means to help smooth introduction to awake conditions on echocardiography and in surface electrocardiogram.
ABR(14w)
When select the sound stimulation frequency, it's to be noted that higher frequencies around 35kHz may give rise to large variability of the threshold due to the wide dependencies on strain and age differences. The strict definition and/or description of click sound stimuli are quite difficult due to the procedures itself of producing click sound with ambiguous variability among researchers with their own programs. It should be considered to set up the common description or not to adopt click sound for ABR.

Andrew Blake's picture

Comments to the pipeline:
Week 6:
Sensitisation with OVA:
This has to be performed in the breeding facility (in case of commercial provider for cohorts this might be difficult)
Alum as adjuvant stays in the body and might influence measurements in individual mice differently
We are concerned about early bleeding at the age of six weeks.
The mice are still young (small!), and blood taking is difficult, sample volume is limited
Bleeding at 6 weeks might be difficult to get a permission from the animal welfare legal office (depends on country)
The bleeding might influence later tests (e.g. behavioural tests should be performed with naive animals)
A pilot study for sensitisation and bleeding at six weeks should be performed, before considered in a high-throughput pipeline
Week 14:
Week 14 has too many tests that are performed on an individual mouse. We suggest to move IPGTT to a different week.
We recommend to perform only once anaesthesia in the pipeline and to analyse the mice consecutively in 1.ABR, 2. Dexa, 3. X-ray, (4. OCT if machine is available)
Body composition: we recommend to stay with DEXA as long as the technology is available for all partners.
Week 16
Our Immunology Screen agrees to the importance of the measurement of leukocyte-populations and immunoglobuline levels in blood and blood plasma. Implementation of a robust immunization scheme within a primary-screening pipeline would be attractive, and flow-cytometric measurement of splenocytes at the end of the pipeline would add valuable data.
Urine analysis is in our eyes not yet a standardized method. Collection of suitable urine samples is time consuming. A Pilot might be needed.
We are concerned of having clinical chemistry and haematology data of challenged mice (reaction on OVA-challenge) only. A pilot should clarify the effects on these parameters concerning values and variability.
Histopathology of lung alveolar and surface structure: a separate and time consuming embedding of the tissue is needed for a proper result (20 min per mouse). We do not expect that it is worth the effort for every mutant line.
We propose to establish the embryology pipeline at a central facility.

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