Immunology/Microbiology study using mice
THE UNIVERSITY OF MELBOURNE
ANIMAL ETHICS COMMITTEE
APPLICATION FOR APPROVAL TO USE ANIMALS FOR SCIENTIFIC PURPOSES
Before completing an application, applicants should familiarise themselves with all relevant guidelines and legislation, including the NHMRC Australian code for the care and use of animals for scientific purposes
(2013).
Applicants should visit the OREI website and download the Application Form each time a new application is prepared, in order to ensure the most up-to-date version of the form is used. Applicants should also read the separate Additional Information sheet for specific guidelines on how to complete the application
To ensure that all AEC members are provided with sufficient information to participate effectively in the assessment of the application, all responses should be clear, concise and written in plain English.
*Please save your application as a Word file (not a PDF) and use the Ethics ID number as the file name*
1. ADMINISTRATIVE DETAILS
Project ID and Title:
ID No:
Title:
Contacts:
Example1
Defining the role of the Amyloid Precursor Protein family in motor neuron disease
Name
Project Supervisor Investigator 1
Primary Contact Investigator 1
Department
Pathology
Pathology
2. PROJECT BACKGROUND & AIMS
2.1 Provide a plain English summary of the background to the proposed work
*Please note, terms defined in the Glossary are indicated by a hyperlink.
caused by the progressive death of motor neurons. Motor neurons are the nerve cells that control the muscles
that enable us to move, speak, breathe and swallow. 10-15% of MND cases are familial (inherited)
, and can
have increased expression of the mutant forms of SOD1 show clinical signs similar to those seen in MND
patients. This transgenic SOD mouse is therefore a very good model of MND and allows us to study the
biological mechanisms of MND.
Despite intensive research to understand the molecular mechanisms of MND, the cause of MND remains unclear. Therefore, it is important to identify and study genes/proteins that can modulate the onset and progression of disease in MND.
mouse models. APP is a brain protein that has increased expression in MND patients, as well as in animal models of MND. In these mouse models is was shown that the absence of APP (i.e. APP knockout mice) caused a significant slowing down in disease progression. This was done by mating transgenic SOD mice with
APP knockout mice to generate a SOD transgenic mouse that does not express APP. These studies
demonstrated that the expression of the APP molecule could modify the disease process in MND.
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APP is part of a gene family that includes genes that are similar to APP. These genes are the amyloid
of the other APP-family molecules in MND in order to better understand their role in MND. We want to study the
APLP2 gene since we have shown in other studies that the APLP2 protein can protect brain cells and allows them to survive better following an injury. This project will test the role of APLP2 in MND by crossing APLP2 knockout mice with the SOD transgenic mice in order to make an MND transgenic mouse model that does not express the APLP2 protein.
2.2 Outline the overall aim and individual aims of the proposed work
The overall aim of this project is to examine the effect the APLP2 gene has on disease progression in the transgenic SOD1-G37R mouse model of MND. The aims of this project are:
1. To generate SOD1-G37R:APLP2-/- and SOD1-G37R:APLP2+/-mouse lines.
2. To characterize the clinical phenotypes of SOD1-G37R:APLP2-/- mice by comparing them to SOD1-
G37R:APLP2+/-, SOD1-G37R, APLP2-/- and wild-type mice (C57Bl6) mice.
3. To characterize the biochemical and pathological phenotypes of SOD1-G37R:APLP2-/- mice by comparing them to SOD1-G37R:APLP2+/-, SOD1-G37R, APLP2-/- and wild-type mice (C57Bl6).
2.3 If applicable, provide details of the relationship of the proposed work to other work, e.g. AEC approved research or teaching, and clinical or agricultural activities. Include, as relevant, application ID numbers.
This project is not directly related to any other AEC projects the Project Supervisor is working on.
3. EXPERIMENTAL OR COURSE DESIGN
Provide the details of your proposed research or teaching activity. It should be clear and concise, but must contain enough detail that a proper assessment of the impacts of the protocols/procedures on each animal (or group of animals) can be made.
You should make reference to: the number and types of animals, relative to specific procedures; the details of each protocol/procedure; the details of pain or distress expected to be experienced by the animals; the details of monitoring; and the fate of the animals.
MOUSE STRAINS
SOD1-G37R
SOD1-G37R is a transgenic mouse that overexpresses the human SOD gene with the G37R mutation (amino
acid glycine at position 37 is mutated to an arginine). The G37R mutation causes motor neuron disease (MND) in humans. The SOD-G37R mice exhibit a wide range of signs consistent with MND symptoms, including a decrease in their ability to move at 6 to 8 months of age, tremors at 6 to 8 months of age, weakness of the limbs
(typically only one side is affected) at 6 to 8 months of age, and progressive weight loss (Wong et al 1995,
Neuron 14:1105-16). Reproductive and developmental features are normal.
APLP2
and lifespan of the APLP2+/- and APLP2-/- mouse is normal and they do not require any modified monitoring programs. The behavioural, physiological, reproductive and developmental features are normal. They have no known major abnormalities. They can be up to 10 % smaller in weight than the wild type background strain.
SOD1-G37R:APLP2
The APLP2+/- and APLP2-/- mouse will be crossed with the SOD1-G37R to generate the SOD1-
G37R:APLP2+/- and SOD1-G37R:APLP2-/- lines. This will test how reducing APLP2 gene expression affects disease outcome in the SODG37R motor neuron disease mice. The phenotype of the SOD1-G37R:APLP2+/- and SOD1-G37R:APLP2-/- lines is unknown as they will be novel lines and therefore the mice will be carefully monitored from birth. A mouse passport will be created after the strain has been established for 6 months. If necessary we will consult the Animal Welfare Officer and Animal Facility Manager on the mouse passport. The animals will be monitored daily once the signs/phenotype develops.
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PROCEDURES AND PROTOCOLS
Aim 1. To generate SOD1-G37R:APLP2-/- and SOD1-G37R:APLP2+/-mouse lines
shown below in Figure 1.
APLP2 -/- mice will be mated with SOD1-G37R mice. The SOD1-G37R mice are hemizygous for the transgene so the progeny from this cross will be SOD1-G37R:APLP2+/- and APLP2+/- at 50:50 ratio. The progeny will be ear tagged for identification purposes and tail clipped (2mm long) at weaning age (3 weeks) for PCR analysis of genotype. The ear tag tissue is not suitable for PCR analysis of the genotype since it is impractical to confidently clean the ear tag tool between mice to a level that would avoid possible cross-contamination of the tissue sample between different mice. This could lead to false positive results in the PCR assay since PCR is a very a sensitive technique. Tail clipping is done with a new razor for each mouse and therefore avoids crosscontamination. The SOD1-G37R:APLP2+/- are kept. The APLP2+/- mice are the incorrect genotype and will be killed by cervical dislocation.
The SOD1-G37R:APLP2+/- mice will be mated with APLP2-/-. The following genotypes will be generated:
SOD1-G37R:APLP2-/- , SOD1-G37R:APLP2+/-, APLP2+/-, APLP2-/- at 25% for each genotype. The progeny will be ear tagged for identification purposes and tail clipped (2mm long) at weaning age (3 weeks) for PCR analysis of genotype. As discussed above the ear tag tissue is not suitable for use in the PCR assay due to potential contamination issue. Tissue for genotyping is obtained by tail clipping. The SOD1-G37R:APLP2-/- ,
SOD1-G37R:APLP2+/- and APLP2-/- mice are kept and the APLP2+/- mice are killed by cervical dislocation.
Mating will continue until the necessary numbers of SOD1-G37R:APLP2-/-, SOD1-G37R:APLP2+/- and APLP2-
/- mice are generated.
Figure 1. Expected genotypes and frequencies
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Aim 2: To characterize the clinical phenotypes of SOD1-G37R:APLP2-/-, SOD1-G37R:APLP2+/-, SOD1-
G37R, APLP2-/- and wild-type mice (C57Bl6)
The SOD1-G37R:APLP2-/- (n=24), SOD1-G37R:APLP2+/- (n=24), SOD1-G37R (n=24), APLP2-/- (n=48) and wild-type mice (C57Bl6; n=48) from Aim 1 will be housed and monitored until they develop signs that reach one or more of the severe criteria listed in the intervention criteria sheet (see attached), at which point they will be killed.
It is expected the SOD1-G37R will be killed at around 8 months of age, since this is when they typically develop one or more signs classified as severe on the intervention criteria sheet.
We do not know if removing the APLP2 gene will increase or decrease the lifespan of the SOD1-G37R:APLP2-/- and SOD1-G37R:APLP2+/- mice. If these mice develop signs of MND then monitoring will be increased to daily, and they will be killed when they develop signs that reach one or more of the severe criteria listed in the intervention criteria sheet. If these mice do not develop one or more severe intervention criteria, we will keep them alive for up to 18 months before killing. The same number of C57Bl6 and APLP2-/- mice will be killed at the same time as the SOD1-G37R:APLP2-/- and SOD1-G37R:APLP2+/- mice.
Experimental assessments of live mice
After 12 weeks of age mice will be assessed twice per week for locomotor function using rotarod and stride length tests and neurological function using neurological scoring system. This will be performed at the same time the mice are weighed. Refer to Figure 2 for the experimental timeline.
Figure 2. Experimental Timeline
Locomotor function in the mice: two tests
- Rotarod assay: the mice will be placed on an elevated horizontal rod that rotates at a defined speed
(accelerates from 4 to 40rpm over 3 minutes). The mice will be allowed to walk freely on the rotating rod and the amount of time the mice are able to remain on the rod will be recorded. As locomotor deficit increases, the amount of time the mice remain on the rod decreases. The mice will be left on the rod for a maximum of 300 seconds (i.e for up to an additional 2 minutes after it reaches top speed of 40rpm), in such cases they will be removed and will be recorded as having no detectable locomotor deficit. Mice that cannot continue on the rotarod up to 300 seconds will fall on to a padded base approx. 15 cm below.
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- Stride length test: the hind paws of the mice will be painted with a water soluble non-toxic paint and the mouse will then walk on a sheet of paper through a cardboard tunnel (approximately 50cm long), leaving paw prints on the paper. The progressive change in the distance between the paw prints will be used as a measure of locomotor deficiency.
Neurological Scoring:
Mice will be scored neurologically using a neurological scoring system (recommended by International Prize 4
Life Group, developed by ALS-TDI). The score criteria:
- Score of 0: Full extension of hind legs away from the midline when mouse is suspended by its tail, and mouse can hold this for two seconds, suspended two to three times.
- Score of 1: Collapse or partial collapse of leg extension towards lateral midline (weakness) or trembling of hind legs during tail suspension.
- Score of 2: Toes curl under at least twice during walking of 12 inches, or any part of foot is dragging along cage bottom/table*.
- Score of 3: Rigid paralysis or minimal joint movement, foot not being used for generating forward motion.
- Score of 4: Mouse cannot right itself within 30 seconds after being placed on either side.
*If one hind leg is scored as 2, food pellets are left on bedding. If both hind legs are scored as 2, Nutra-Gel diet will be provided as food in addition to food pellets on bedding and a long sipper tube is placed on the water bottle.
Aim 3: To characterize the biochemical and pathological phenotypes of SOD1-G37R:APLP2-/-, SOD1-
G37R:APLP2+/-, SOD1-G37R, APLP2-/- and wild-type mice (C57Bl6) mice
The mice will be kept alive until they develop severe signs that require intervention, as listed on the intervention criteria sheet, at which point they will be killed and the tissues will be removed and used for biochemical and histological analysis.
KILLING OF MICE
Mice with the incorrect genotypes will be killed by cervical dislocation.
Mice of the correct genotypes that reach a particular timepoint or display signs classified as severe on the intervention criteria sheet that requires them to be killed, will be anaesthetised and intracardially perfused. The
ketamine (120mg/kg body weight) in neutral saline solution using a 26G needle. Once the mice are unconscious
(this is tested by pinching the toes and looking for withdrawal reflex) the chest cavity will be opened and the aorta cut. A 23G needle, attached to tubing which feeds into a reservoir of cold PBS, will be inserted into the left ventricle of the heart. Using gentle pressure or a dialysis pump, cold saline solution will be perfused into the mouse for 2 to 3 minutes followed by infusion of cold 4% paraformaldehyde in 0.1M phosphate buffer containing
0.5% NaCl for 1 min. This will kill the mouse, and allow for the preservation of tissues for biochemical and histological analysis.
PAIN AND DISTRESS
Genotyping and Tagging:
The SOD1-G37R:APLP2-/-, SOD1-G37R:APLP2+/- , APLP2-/- and SOD1-G37R mice will be identified by eartagging, and a tail snip (2-3 mm long) at weaning age (3 weeks) will be used for PCR analysis of genotype. As discussed above the ear tag tissue is not suitable for use in the PCR assay due to potential contamination issue.
Tissue for genotyping is obtained by tail clipping. These procedures will be performed once and will cause minimal pain and distress to the mice.
Killing by Cervical Dislocation:
Mice of the incorrect genotype will be killed by cervical dislocation. This procedure will cause minimal distress or pain.
Killing by anaesthesia and perfusion:
Prior to perfusion the animals will be anaesthetised. The xylazine/ketamine will be prepared in neutral saline solution and administered IP. The xylazine/ketamine injection will cause minimal pain and distress to the mice.
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Body weight monitoring:
Mice will be taken from their cages and placed briefly on a set of scales. This will cause minimal pain and distress to the mice.
Mice locomotor function:
Both the rotarod assay and stride length test (as described above) will cause minimal pain and distress to the mice. The mice do not display signs or pain or distress during these tests. For the rotarod assay the distance of fall is approx. 15 cm and is on to a padded base in order to prevent injury and minimize stress.
Neurological Scoring:
Mice will be suspended by the tail to observe the extension of hind legs. This will cause minimal pain and distress to the mice.
MONITORING
General housing conditions (availability of food, water, cleanliness) will be checked daily.
Mice will be monitored for body weight and signs of MND assessed against the intervention criteria checklist by visual monitoring of appearance, behaviour, activity & response to provocation, body condition score, decreased appetite or drinking, interference with mobility/movement/gait, breathing, status of eyes and nose, faeces and vocalisation.
- SOD1-G37R, APLP2-/- and wild-type mice (C57Bl6) will be weighed and monitored weekly.
- SOD1-G37R:APLP2-/- and SOD1-G37R:APLP2+/- mice will be weighed and monitored daily in their first 4 weeks of life. If there is no significant change in the phenotype or mice body weight after 4 weeks of age, monitoring will be reduced to three times per week.
- For SOD1-G37R:APLP2-/-, SOD1-G37R:APLP2+/- and SOD1-G37R mice, the frequency of monitoring will be increased to daily as signs of MND become more progressive, as assessed against the intervention criteria sheet.
Recorded information from monitoring of the mice, together with body weight, neurological scoring and locomotor function, will define the clinical stage the mice have reached as per the monitoring and intervention sheets. When an animal records one or more of the signs classified as severe on the intervention criteria sheet, the mouse will be anaesthetised with xylazine/ketamine and killed by intracardial perfusion.
The animals will be monitored by the Animal Facility staff, Investigator 1, Investigator 2, Investigator 3 and
Investigator 4. New investigators who require training in monitoring will not monitor animals independently until fully trained and deemed competent by the Project Supervisor.
FATE OF THE ANIMALS
All the animals used in this study will be killed. Killing will be by either cervical dislocation (animals of the wrong genotype) or anaesthesia plus perfusion with paraformaldehyde.
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4. INVESTIGATORS & COMPETENCY
In the table below, identify individual investigators against the protocols/procedures that they will be performing. For each protocol/procedure, indicate whether the investigator is ‘C’ (competent) or ‘T’
(needs training). Where an investigator is not performing a particular protocol/procedure, the associated cell should be left blank. All Animal Facility Managers need to be named here if animals are to be located in University animal facility premises. In addition, animal facility staff must be included if they will be involved in performing procedures.
PROTOCOL/PROCEDURE
Investigator 1
Investigator 2
Investigator 3
Investigator 4
Animal Facility staff
Animal Facility
Manager
T
T
C
C
C
N/A
T
C
N/A
T
C
N/A
T
C
N/A
T
C
N/A
T
C
C
N/A
T
C
C
N/A
T
C
C
C
N/A
T
C
C
N/A
5. HOUSING OF ANIMALS
5.1 For each species/strain requested, complete a row in the table.
Species / Strain
All mice
Location
Pathology and Anatomy & Cell Biology
Animal Facility
Housing
Standard
Grouping
Grouped
5.2 If relevant, provide further details of housing, including, as relevant, details of outdoor housing, any special housing requirements, details of enrichment, etc. Where housing is not applicable, please explain why.
Not relevant.
6. REPLACEMENT
Replacement refers to methods that avoid or replace the use of animals.
6.1 Explain why it is necessary to use animals for the proposed work.
These experiments involve the assessment of life span, motor function and neurological deficits to try to identify some of the underlying mechanisms of MND. A physiologically relevant mammalian disease phenotype for MND is therefore required, and this can only be achieved using a live animal model.
6.2 Provide evidence for the consideration of alternatives to animal use.
Due to the complex biological nature of this project, the experiments cannot be simulated or modelled in any way. Furthermore, while cell culture can be used for some simple physiological studies, the assessment of life span, motor function and neurological deficits, as required for this project, cannot be achieved in cell culture systems.
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6.3 Provide justification for the choice of animal/s (species, strain, genetic modification, sex and age)
Mice are chosen since the transgenic SODG37R (MND) model and the APLP2 knockout animals are only available in mice. The SODG37R mice are a strong model of the human disease and display the motor dysfunction as well as the reduced lifespan that occurs in human motor neuron disease. We are studying both male and female animals to ensure gender balance. Age reflects the time-points the disease and disease endpoints occur.
7. REDUCTION
Reduction refers to methods that minimise the number of animals required to achieve the work’s aims.
7.1 Provide statistical or other justification for the number of animals requested. Break down the total number by procedures, treatments, repeats, groups, etc.
ANIMAL NUMBERS - JUSTIFICATION
The number of animals requested for the experimental component of the study is based on the numbers required to achieve statistical significance and reflects the numbers used in previous MND studies for preclinical animal research (Ludolph et al., 2010) and those used by co-investigator 2. For a survival study Ludolph et al.,
2010 recommends the use of n=12 mice per gender per group. The Ludolph et al paper is the published guidelines from the European ALS/MND meeting in 2009 that met to establish the guidelines for performing
“best practice” preclinical animal research in MND.
For each genotype the following mice numbers will be killed when they develop signs that reach one or more of the criteria listed in the severe intervention criteria.
SOD1-G37R (n=12 female, n=12 male)
SOD1-G37R: APLP2-/- (n=12 female, n=12 male)
SOD1-G37R: APLP2-/+ (n=12 female, n=12 male)
APLP2-/- (n=12 female, n=12 male) x 2 groups
C57Bl6 (n=12 female, n=12 male) x 2 groups
The wild-type (C57Bl6) and APLP2-/- group will be double in size in case the SOD1-G37R:APLP2-/- or SOD1-
G37R:APLP2+/- die at a different age to the SOD1-G37R. This will allow the study to provide a relevant age control group to compare for both the SOD1-G37R as well as the SOD1-G37R:APLP2-/- and SOD1-
G37R:APLP2+/- mice.
Breeding Component:
The number of mice required to generate the SOD1-G37R:APLP2-/- and SOD1-G37R:APLP2+/- lines requires an estimation since we do not know the exact size of each litter and in particular how many mice of the incorrect genotype will be born and are then required to be killed. Our mating scheme indicates 50% of the litter will be of the incorrect genotype. We have calculated we need 10 SOD1-G37R and 30 APLP2-/- mice for the breeding to generate the necessary SOD1-G37R:APLP2-/- and SOD1-G37R:APLP2+/- mice for the experimental component (Reference: Ludolph et al. Amyotrophic Lateral Sclerosis. 2010; 11: 38-45). This is assuming litter sizes of 4 pups, and each female has 2 litters. For 15 female mice of a genotype = 15 females x 2 litters x 4 pups = 120 pups. 50% correct =60 pups. The same applies for the F2 breeding. These are conservative calculations and assume small litter sizes. Breeding will stop once the reuired number of mice for each genotype is reached.
7.2 Have the animals been used in another project? If so, outline the cumulative burden and provide justification for re-use.
No.
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8. REFINEMENT
How have techniques been refined to minimise impacts on animal welfare?
All procedures have been refined throughout previous projects to cause minimal pain and distress to the animals. The animals will be closely monitored to detect disease progression and ensure animals are killed at the appropriate intervention point.
9. OVERALL JUSTIFICATION
Explain how the potential impacts on the wellbeing of animals in this project are justified by the potential benefits of the proposed work.
Motor Neuron Disease (MND) is a fatal human disease and therefore it is important to understand the cause and molecular pathways that modulate this disease. The APP gene has been shown to have an important effect in modulating MND. To properly understand the role of the APP-family in MND, it is necessary to investigate the effect that the other APP-family genes have in MND. In this project we will investigate APLP2. If APLP2 can modulate disease progression then this will identify new disease pathways and novel drug targets and will guide the development of new treatment options for MND.
10. GLOSSARY
Provide descriptions/definitions of abbreviations and scientific terms in language that can easily be understood by the lay reader.
Scientific Term
Motor neuron disease (MND)
Neurodegenerative
G37R
Familial
Transgenic
APP
APLP2
Allele
Knockout mouse
Hemizygous
Intra-peritoneal
Superoxide dismutase-1 (SOD1)
Lay Description
Motor neuron disease: a human disease in which motor neurons undergo degeneration and die.
Associated with the deterioration of the nervous system.
Superoxide dismutase-1 is a protein that binds copper and zinc ions, and is responsible for protecting cells against oxidative damage.
G37R is a mutation that occurs in the SOD-1 gene that causes motor neuron disease (MND) in humans.
A condition that tends to occur among members of the family than by chance alone, the etiology is either genetic or environmental or can be a combination of the two.
Containing a gene or genes transferred from another species
Amyloid Precursor Protein, a brain protein whose expression is highly increased in MND patients and in animal models of MND.
Amyloid Precursor Like Protein 2, a homologue of APP, a protein that can modulate the survival of neurons against neuronal damage.
A genetically modified mouse where a specific gene has been engineered so that its expression is repressed resulting in a lack of protein expression.
One of two or more versions of a gene. An individual inherits two alleles for each gene, one from each parent.
One copy of a gene, with no corresponding copy on the other chromosome.
Located in the belly region of the mouse, site for the anaesthetic injection.
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