FELASA unsure Flashcards
Explain different ways of genetically modifying an animal and give examples of genetically modified animal models and their use in biomedical research.
Spontaneous mutants
Spontaneous mutants are animals characterized by a mutation which occurred spontaneously and has proven to serve a beneficial scientific purpose.
Examples:
Nu mutation in Foxn1 gene (Foxn1nu mice): nude, thymus lacking mice, which lack T-cells and thus are unable to reject transplants.
SCID mutation in the Prkdc gene of Severe Combined Immune Deficiency (SCID) mice leads to the generation of mice lacking both T and B cells.
Mice with a spontaneously occurred leptin deficiency are prone to the development of obesity and diabetes.
Selective inbreeding
Selective inbreeding is a kind of artificial selection and refers to the process of humans selecting which animals to mate to produce offspring with scientifically desirable/relevant characteristics.
Examples:
Spontaneously hypertensive rats (SHR)
Non-obese diabetic (NOD) mice
Genetically modified animals
Transgenic animals
Transgenic animals are animals the genome of which has been manipulated by insertion of foreign genomic material. Introduction of novel genes works through both heterozygous and homozygous expression.
Knockouts
Knockout animals are animals in which a gene has been removed or made non-functional. Knockout only works if the expression of both copies of a gene is impaired, that is homozygotic removal/impairment of function is required.
Knockins
Knockin animals are animals in which a gene from a foreign species is inserted in the same site as the same gene in the homologue version (i.e., a one-for-one substitution of genomic information). Homozygous expression is required for knockin of genes to work.
Examples of genetically modified animals:
Rag1 mutants are analogous to spontaneous SCID mice, which lack both T and B cells.
Cystic fibrosis models can be generated via creation of knockin mice, where the murine Cftr gene is swapped with one of the human mutations (e.g., ∆F508).
Creation of fluorescent animals, tissues, or cell-populations within live animals.
R6/1 mice = mice spliced with the human base pair repeats causing Huntington’s chorea.
Methods of genetic engineering Pronucleus microinjection One pronucleus of a fertilized egg, which contains 2 pronuclei (one from each parent), from a mated female is injected with a DNA construct containing a desired gene. Often, DNA is injected into the male pronucleus. Genetically manipulated eggs are transplanted into a pseduopregnant female, which gives rise to heterozygous off-spring, as only one pro-nucleus is injected. Breeding for a couple generations is thus required to achieve homozygosity. Insertion of the genetic construct into the genome of the pronucleus is random, and this method is thus applicable only for the random insertion of a functional transgene, not the knockin nor the knockout of specific genes. This method is applicable to all species.
Embryonic stem cell transfection Embryonic stem cells (ECs) are obtainable from blastocysts of mice, rats, and in principle humans, and their genomic contents can be manipulated using all available gene modification techniques. Upon genetic manipulation and selection, ECs are reintroduced into another blastocyst, which is then injected into an anaesthetised, pseduopregnant female, from which the injected blastocyst originated. The resulting offspring is chimeric, meaning that they consist of both wild type cells and gene modified cells. From this initial generation of chimeras, homozygotic gene modified mice can be obtained through breeding of multiple generations: chimeric mouse is mated with a wildtype mouse, which hopefully gives rise to off-spring of both sexes, both containing gene modified germ cells. These mice are then mated to generate homozygotic mice. Hopefully, both sexes are represented, so it is possible to continue breeding of homozygotic mice.
Cloning and nuclear transfer
A cell (e.g., a fibroblast) is isolated from an animal and cultivated, and its genomic material is manipulated with the desired changes. Then, the nucleus of a sow’s egg is replaced by a gene modified nucleus. After further development to a blastocyst, the gene modified embryo is transferred to the uterus of a pseduopregnant sow, which will result in the birth of a gene modified piglet.
This approach is applicable to all species, and can be used for both insertion of functional transgene, and as it offers spatial control, it can also be used for creation of knockin and knockout animals.
Nuclease based genome manipulation
Multiple nuclease-based genome manipulation tools exist:
Zinc-finger nucleases
TALENs
CRISPR/Cas9
Conditional gene modification
Conditional gene modifications are gene modifications which are either done in specific tissues or for limited time periods only.
Conditional gene modification is obtainable using the cre-lox system, which is enacted through a recombinase, Cre, which can cut the DNA string at specific LoxP cutting sites, and either flip or excise floxed sequences, depending on the orientation and number of LoxP sites.
Both LoxP sites and Cre recombinase must be present in the same cells at the same time for the system to work. For example, knockout of a gene in a specific tissue can be achieved by introducing LoxP sites floxing the gene of interest in one animal, for example through embryonic stem cell transfection or nuclease-mediated genome manipulation techniques. In another animal, a construct encoding the Cre recombinase and a tissue-specific promotor is inserted into the genome by one of the techniques allowing targeted insertion. If these genetically modified mice the Floxed GOI mouse and the tissue specific Cre mouse, are now mated, mice which are knockouted for the GOI in a tissue specific manner are generated as a result.
More complicated variants of the Cre-Lox system expand its applicability.
Define and discuss the concept of a humane end-point and give some examples on humane endpoints. Discuss different ways of assessing suffering in animals.
Two distinct types of endpoints exist:
Experimental endpoints = predefined endpoint of the experiment, where all necessary data have been obtained.
Humane endpoints = the earliest point at which an experiment can or should be terminated, with the purpose of eliminating unnecessary suffering.
Humane endpoints are identified by assessment of early behavioral and clinical signs in combination with model-specific signs, to introduce as early endpoints as possible. Endpoints therefore differ depending on the severity of the experiment, of which 4 classes exist:
Non-recovery = procedures which are performed entirely under general anesthesia from which the animal shall not recover
Mild = procedures from which the animals are likely to experience short-term mild pain, suffering or distress, as well as procedures with no significant impairment of the well-being or general condition of the animals.
o Example: administration of anesthesia
Moderate = procedures from which the animals are likely to experience short-term moderate pain, suffering or distress, or long-lasting mild pain, suffering, or distress as well as procedures likely to cause moderate impairment of the well-being or general condition of the animals.
o Example: surgery under general anesthesia
Severe = procedures from which the animals are likely to experience severe pain, suffering, or distress, or long-lasting moderate pain, suffering, or distress as well as procedures likely to cause severe impairment of the well-being or general conditions of the animal.
o Example: severe restriction of movement over a prolonged period.
Thus, humane endpoints are present to minimize potential suffering and should be chosen in relation to the severity of the experiment. Humane endpoints are often defined in welfare protocols to ensure easy detection of their occurrence. Parameters defining humane endpoints are like those used in pain assessment and should be obtainable through cage side monitoring. Death is NOT ideal as humane endpoint (too extreme)!! An example of a more extreme humane endpoint is 20% body weight loss.
Pain and stress are very closely linked, as noxious stimuli will initiate a stress response. To achieve control of pain and stress, the following is required:
– Recognition
– Assessment
– Alleviation
It is important to recognize signs of pain and distress early on (you don’t want to end up in a situation where pain recognition is easy!), as a suffering animal in severe distress must be euthanized immediately.
Different parameters are used for pain and stress assessment
Physiological parameters (difficult to measure in smaller animals)
o Heart rate
o Blood pressure
o Body temperature
Biomarkers
o Corticosteroids (increases during stress)
o IgA (decreases during chronic stress)
o Noradrenaline or adrenaline in blood
o C-fos activation
o Oxidative stress markers
o “emotional” stress markers (8-OH-DPAT)
Behavioural and clinical signs
o Can be assessed either subjectively (not as accurate as objective assessment) or objectively (quantifiable). Generally, objective scoring of pain related behaviour is preferable over subjective scoring (greater precission).
o Behavioural readouts can be affected by drugs and time of day of the assessment (due to hormone level fluctuations). These parameters must thus be considered during pain assessment.
o General signs to look for (differs greatly among species – normal behaviour must be known in detail for assessment to be meaningful):
* Is the animal alert?
* Normal movement?
* Does it eat or drink as usually?
* Is it protecting a certain part of the body?
* Does it sound when being handled?
* Does it bite or lick itself more than normal?
* Does the animal exhibit abnormal posture?
* In rats: chromodacryorrea (= red coloured tears)
o Pain related behaviours (rats)
* stereotypic behaviour, which is used as a coping-mechanism by the animals.
* Twitching (often seen with a hop either preceding or following the twitch)
* Back-arching
* Falling
* Abdominal press
* Writhing
* Staggering (= vaklende gang)
* Facial expressions (grimace scale)
o Clinical signs
* Body weight loss
* Food and water consumption (decreases –> anhedonia)
* Urination and defecation (increases –> diarrhoea)
* Fur quality (decreases)
Explain and discuss basic procedures and principles for experimental surgery including e.g. principles and methods of aseptic technique, suturing techniques, tissue handling and strategies for minimizing inflammation, infection and post-operative pain.
Aseptic techniques
o Aseptic techniques can be defined as methods and practices that prevent cross contamination during surgery, which involves proper preparation of the facilities and environment, surgical site, surgeon, and the surgical equipment.
o The purpose of aseptic techniques is to limit the animal’s exposure to both endogenous and exogenous microorganisms and no pathogen has yet developed resistance to aseptic techniques.
o The concept of working aseptically involve sterile things only touching other sterile items and unsterile things only touching other unsterile items, thus keeping sterile and unsterile items separated.
Tissue handling should follow Halsted’s principles:
o Asepsis
o Gentle handling
o Hemostasis (stopping bleeding)
o Closure of dead space
o Careful approximation of tissues
o Avoidance of tension
o Minimization of foreign materials
Working in accordance with Halsted’s principles requires proper training and selection of appropriate instruments, as instruments are designed to prevent undue trauma and aid in gentle handling.
Wound closure and suturing
o Elimination of dead spaces is important
o Sutures in inner layers (muscles, fascia) must be tied tightly
o Sutures in skin must be less tight to avoid induction of inflammation and pain.
o The correct suture material must be chosen, as different materials elicit distinct tissue reactions (use synthetic material is possible!). Tissue absorbability of the chosen materials must also be considered.
o Different suture techniques exist:
* Simple interrupted sutures
Time consuming
Secure; loss of one suture does not open entire wound
* Continuous sutures
Easy and quick
Better seal of wound edges
Less secure
Examples of experimental surgical techniques include:
o Laparotomy = abdominal surgery
* Various approaches (midline, flank, paramedial, transverse) exist depending on organs of interest. Medial incision along linea alba is least traumatic.
* Necessary incisions must be made large enough to improve accessibility and prevent/limit undue trauma.
o Thoracotomy = thoracic surgery
* Obtainable through intercostal space or via median sternotomy
* Requires ventilator
o Catheterization = the insertion of a catheter into a body cavity (e.g., venous catheter, urine catheter etc.).
o Craniotomy = the removal of parts of the skull to access the brain
Lab animal feeding and nutrition: Describe various types of diets and feeding schedules and discuss the possible effects in animal studies.
Laboratory animal diets can be broken into nutrient fractions
o Macronutrients (burnable)
* Carbohydrates
Digestible forms
Indigestible forms = fibers
* Protein
* Fat
o Minerals (un-burnable)
o Vitamins
* Fat soluble: A, D, E, and K
* Water soluble: B vitamins, C vitamin (only essential in guinea pigs and primates, as other species can produce it themselves.
Only a fraction of the energy intake is available as metabolizable energy. Indigestible contents pass through the gut and leave the animal as faecal energy, while some digestible energy is excreted through urine.
Metabolizable energy must be distributed between catabolic (break-down of larger molecules into smaller ones), energy releasing processes, and anabolic (building larger molecules from smaller constituents), energy consuming processes.
Diets must be regulated based on the current state of the animal, e.g., growing, pregnant, and lactating animals need more energy. As animals differ in how much of their GI-tract is constituted by small or large intestine, respectively, diet composition must reflect these differences (protein vs. fiber contents).
Diets for laboratory animals are produced by commercial laboratory animal diet producers, and complete diet fulfilling all nutrient requirement for each species in different life stages are available. Some are based on natural ingredients, referred to as natural ingredient diets or chow, while diets based on synthetic compounds are called purified diets.
Natural ingredient diets
o Natural ingredients include whole grains (corn, barley, and wheat), mill by-products (bran), high protein meals (soybean, fish, casein) and mineral sources (bone meal). Mineral and vitamin mixtures are added as well.
o Both breeding and maintenance natural ingredient diets, which vary in their nutrient composition, exist.
o Natural ingredient diets exist as powders, pellets, extrudates and hybrid pellets.
* Pellets are not cooked during manufacturing and the ingredients are thus raw when fed to animals, which causes a low digestibility associated with increased intake amounts. The optimal/preferred pellet size differs across species.
* Extrudates are cooked during manufacturing, which increases the digestibility, and animals thus must eat less to fulfil nutrient requirements. Extrudates are usually more expensive than pellets.
o Natural ingredient diets can be made using either fixed or variable formulas:
* In fixed formula natural ingredient diets, a fixed amount of each ingredient is mixed into the diet, which means that the level of nutrients will vary, as nutrient levels vary in natural ingredients.
* In variable formula natural ingredient diets, a variable amount of ingredients is mixed into the diet, with the aim of standardizing/fixing nutrient levels in the diet.
Advantages and disadvantages of natural ingredient diets
Advantages
o Cheap
o Palatable (liked by animals)
o Nutritious
Disadvantages
o Variation between batches
o Single components are non-removable
o Contains uncontrolled biologically active compounds (* This can also be an advantage, as these compounds are needed by certain models.)
o Contamination risk, as most components originate from fields which might be inhabited by other animals.
Purified diets
o Different molecules constitute the macronutrients of purified diets:
* Protein
Casein, simple amino acids
* Carbohydrates
Sucrose and starch
Fibers: cellulose
* Fat
Corn oil
Mineral and vitamin mixtures are added as well.
o Purified diets are mostly used for specific research purposes. It is important to match control diet and experimental diet when making diets deficient of one single component.
o Purified diets are often artificially coloured to ensure correct feeding. However, this might be a potential source of bias, as blinding is impaired if it is known what group is fed what diet.
Advantages and disadvantages of natural ingredient diets
Advantages
o Standardized (low variation between batches)
o Single components removable
Disadvantages
o Aversive
o Expensive
o Lack biologically active compounds of importance for specific models
Feeding schedules:
o Ad libitum feeding, meaning that food is always available, is the most common way to feed rodents
o Restricted feeding means that animals are given exactly the amount of food needed to secure intake in accordance with daily nutrient requirements every day.
o Gavage (= oral dosing with a gastric tube) removes oral digestion, and data thus might be less translational compared to humans. However, it is more precise and individual compared to dosing through the diet or drinking water.
o Pair feeding is used to obtain two groups eating the exact same amount: the food intake in one group is registered, and the same amount of food is supplied to the other group.
The impact of different feeding schedules and diets:
o Ad libitum fed animals have a higher growth rate than restrictedly fed animals. However, the expected life span of ad libitum fed animals is lower than that of restrictedly fed animals, as ad libitum fed animals have a higher disease rate than restrictedly fed animals.
o Fasting of mice can have significant impact on experimental results, as fasting induces a state of topor in rodents, a sort of pseudohibernation during which rodents reduce their metabolism and body temperature due to lack of food, which appears in bouts. This is observed as changes in body temperature, corticosterone levels, most blood parameters, and body weight.
o Diet affects the microbiota. Therefore, animals of a study should be fed from the same batch of the same diet throughout a study.
Discuss spontaneous infections in lab animals, their importance for animal experimentation and ways to prevent spontaneous infections
Diseases in laboratory animals which arise spontaneously are often infectious, meaning that they arise from infection by viruses, bacteria, and parasites. These diseases can affect experimental animals to different extents and examples include:
o Hepatitis in susceptible mice and rat strains caused by coronavirus infection.
o Tyzzer’s disease caused by Clostridium piliforme infection of both mice and rats.
o Glässer’s disease caused by Haemophilus parasuis infection in pigs.
Usually, spontaneous infections are unwanted, as microorganisms in laboratory animals may be zoonotic, cause disease in the animals, or otherwise interfere with research. However, some microorganisms may be important for the induction of specific animal models, and presence of microorganisms might improve translatability.
Diseases can be either fulminant, i.e., causing observable clinical symptoms, or latent, which refers to diseases with no clinical expression/manifestation.
Infections can affect animal research both directly and indirectly:
o Direct influences
* Immunological modulation
* Physiological modulation
* Oncological modulation
* Fertility modulation
* Microbiological competition (compete with experimental infections)
o Indirect influences
* Contamination of biological products
* Activation of direct effects
Protection against infections in laboratory animals in accordance with EU guidelines is achieved using a three-step rocket:
o Rederivation
* C-section
* Embryo transfer
o Protection
* Barrier housing (instead of conventional housing)
* Isolator
o Continuous health monitoring
Furthermore, prevention of infection from contaminated biological materials used in research through proper screening and cleaning is important.
Describe various factors to be considered when caring for an animal after surgery. Discuss different options for pain treatment and the advantages and disadvantages of different protocols for pain treatment, discuss when and how to use antibiotics and provide an overview of general factors relevant for smooth post-operative recovery of laboratory animals such as pigs and rodents
A
Careful pre-procedure planning, which includes preparations before anaesthesia, which can be either local or general, and choice of anaesthetic regiment, is essential to spare animals as much pain as possible. The purpose of general anesthesia is to bring the animal in a state which allows the performance of surgery or any other procedure, without causing the animal to suffer, and reversibility and controllability constitute the basic elements of general anesthesia
The anaesthetic triad is composed by:
loss of consciousness (anaesthesia)
no feeling of pain (analgesia/anti-nociception)
muscle relaxation.
Multiple drugs administered in combination are usually needed to fulfil the criteria of the anaesthetic triad, which is referred to as balanced anaesthesia and constitutes the predominantly used anaesthetic regimen.
Lower dose of each drug can be given
* Minimization of the side effects from each drug
Multiple factors must be considered prior to surgery, to ensure smooth postoperative recovery of experimental animals:
o Evaluation of the animal
o Practical considerations
* Is assistance needed?
* Does the animal have to be fasted or not? Larger animals are usually fasted, however, it is not required in rodents, as these species cannot vomit (due to presence of prominent cardiac sphincter).
o Pre-anesthetic medication
o Choice of anesthetic drug/regimen (single vs. combinations of drugs – balanced anesthesia is the strategy most often used due to beneficial synergistic effects).
* Objective of the experiment
Human or animal patient: make sure the animal survives in a good condition:
Experimental animals: make sure the scientific data collection is as accurate as possible. Interactions of anesthetics with experimental data can be resolved by:
Studying the literature
Consulting experts and discuss with colleagues
Investigate if and how the model is affected (i.e., make pilot studies).
* The surgical procedure
* The estimated time of surgery
* Species
* Inhalation vs. injection
Anesthetic depth is more easily controlled using inhalations anesthesia.
Routes of administration for injection anesthetic:
subcutaneous (most optimal, choose when applicable)
intraperitoneal (should be avoided due to risk of damaging internal organs)
Intramuscular (rarely used).
o Monitoring of anesthesia and plan for action
* Absence of reflexes
Withdrawal reflex (must be checked regularly throughout surgery)
Palpebral reflex (relevant for larger animals)
Righting reflex (= a sequence of coordinated movements involving head and limb rotation such that the animal lands on its feet. Absence must be checked before surgery)
Corneal reflex (relevant for larger animals)
* Circulatory functions (heart rate/pulse, blood pressure)
* Respiratory functions
* Body temperature
o Analgesia
* Pre-emptive, intra- or postoperative
o Postoperative supervision and treatment
The postoperative needs of animals differ among species and can include:
Postoperative pain
o Provide analgesia (however, it must be taken into consideration that analgesic drugs might affect several physiological, pharmacological, and endocrine systems, and consequently the experimental data (depends on the model and the analgesic in use). However, this is not always the case, and experiments must thus be conducted. A balance thus exist between the effects of analgesic drugs and the pain and stress induced by their absence, which both affect physiology.
– Dehydration
o Body warm saline IV or SC during anesthesia and/or postoperatively
o Provide easy access to drinking water
– Hypothermia
o Heating blankets (larger animals), infrared heating lamps or incubators (rodents)
o Body temperature must be maintained until animal is fully recovered
o Be aware of overheating
– Infections
o Asepsis
o Observe wound healing for necrosis or infection.
o Provide antibiotics if necessary.
Antibiotics are not to be seen as a substitute for proper aseptic technique but are necessary in some instances (antibiotics do not work against viral, fungal or parasitic infections). Generally, broad spectrum antibiotics are instituted before surgery and continued for 2-3 days. However, use of antibiotics contributes to development of resistance and also impacts the microbiome of the experimental animals, which might affect experimental results. Thus, antibiotics, if strictly necessary, should be administered to both control and experimental animals to avoid antibiotics constituting a confounding factor.
– Postanesthetic restlessness and self injury (can be caused by pain or severe distress)
o Consult veterinarian
o Use sedatives or analgesics if indicated
For animal models for which pain relieve is not feasible (e.g., in pain models), other means of refinement must be used. Multiple examples of non-pharmacological refinement approaches exist;
Introduce early experimental endpoints to avoid unnecessary suffering
Develop strictly defined humane endpoints with subsequent actions specified
If animals are clearly affected within approved severity, facility the situation by easy access to food and water, comfortable bedding, etc.
Give examples of occupational hazards in laboratory animal facilities and discuss how to minimize these potential risks
Occupational hazards in laboratory animal facilities include
– Allergies (which can potentially develop into asthma)
Various allergens from mice, rats, guinea pigs, rabbits, cats, and dogs are the most common source of allergy. These allergens are proteins, and originate from different sources (e.g., hair, dander, urine, serum, or saliva). Exposure to allergens can happen via skin contact, inhalation, or by injection accidents. The average time to develop symptoms of allergy caries between 2-8 years from initial exposure.
– Physical injuries
o Bites and scratches (usually caused by smaller animals)
o Kicks and butting (usually caused by larger animals)
o Accidental punctures due to incorrect handling of sharp objects such as needles, scalpels, or glass pipettes.
– Harmful substances
o Biologicals
* Infectious agents (e.g., viruses)
* Cells from other species
* Toxins
o Radioactive isotopes
o Adjuvants (= powerful immune stimulants used to promote antibody production)
o Chemicals
* Dangerous chemicals (e.g., carcinogens, mutagens, reproductive toxicants, etc.)
* Inhalation anaesthetics (e.g., isoflurane)
* Pressure bottles (e.g., oxygen, nitrogen, carbon dioxide)
– Zoonoses
Zoonoses are bacterial, viral, fungal or parasitic infections which can infect both animals and humans. Zoonoses can arise from animals (primates, dogs, cats, rodents, pigs), cell lines, tissue cultures, transplants, and biological products. Infection can occur by multiple routes (ingestion, inhalation, inoculation, contamination of skin and mucous membranes).
How to minimize the potential risk of occupational hazards in laboratory animal facilities:
– Allergies
o Keep levels of allergens in the environment as low as possible (e.g., by keeping animal density down, using the correct bedding material, through effective and sage cleaning, monitoring allergen levels etc.).
o Use personal protective equipment (protective clothing, face masks, gloves, particle-filtering respirators).
o Do not carry allergens with you when leaving the animal facility
o Limit work with live animals
– Physical injuries
o Learn how to handle animals and use proper restraint
o Hand sharp objects with care and use them properly
– Harmful substances
o Biologicals
* Adding warning label on cage
* Only opening cage in biosafety cabinet or hood
* Wearing protective clothing: gloves, eyewear, mask, coverall
* Good needle practice
* Disinfection/autoclaving of waste
o Radioactive substances
* Follow legal requirements for room and laboratories, and education of personnel
* Have expertise available
* Keep and transport radioactive substances safely
* Prepare syringes before experiments
* Restrain animals properly
* Inject in safety cabinet
* Use personal protection
* Keep animals enclosed
* Handle waste properly
* Clean up after work procedures
o Adjuvants
* Prepare individual syringes for each animal in advance of animal handling
* Perform work in safety cabinet
* Wear personal protective equipment
* Make sure the animal is restrained properly (anesthesia could be indicated)
* Practice correct disposal of used materials such as syringes and needles
– Chemicals (dangerous chemicals, inhalation anaesthetics, pressure bottles)
o Know the hazardous properties of the chemicals in use
o Take precautions during preparation, injection etc. (e.g., use proper ventilation)
o Isolate animals
o Take precautions during waste handling
o Use proper personal protective equipment
o Restrain compressed gas cylinders securely
– Zoonoses (rare due to strict health assessment regimens utilized at animal facilities today)
o Work with microbiologically defined animals
o Use only biological products from safe sources
o Use barrier units in the animal facility
o Have good working routines
o Use adequate personal protective equipment
o Take proper action in case of an accident
