Experimental Animal Models Flashcards
Methods of developing animal models
- Spontaneously occurring variations
- Selective breeding
- Genetic modifications
- Drug effects
- Environmental effects
- Surgical & physical alterations
What were the earliest animal models available?
Spontaneously occurring variations within the animal population
What does selective breeding allow?
To keep a trait within a population
Examples of breeding colonies
- Leptin deficient mouse - occurred spontaneously, mice develop symptoms similar to obesity & T2DM
- SHR - selective breeding of WK rats which naturally have HBP
- Nude mouse (athymic mice) - immunocompromised
How is an animal model created by drug effects?
An otherwise healthy animal is treated with a compound known to induce a disease condition or symptoms that closely mimic the disease
Common way to model depression
Chronic mild stress test - animals are subjected to unpredictable stressors resulting in a variety of behavioural deficits: food & water deprivation, small temp reductions, changes of cage mates, changes in light/dark cycles
Examples of drug-induced animal models of disease
- Streptozotocin induced diabetes in rats - IV injection, makes pancreas swell & causes degeneration of islet of Langerhans’ beta cells
- Deoxycorticosterone induced HT in rats, dogs, pigs - prolonged mineralocorticoid admin
- MPTP for PD in primates, nice - admin triggers rapid destruction of DA-synthesising neurons in SN
What is a common model of early life stress?
Maternal separation model - pups separated from mother for several hrs per day over a period i.e. 3hr/day during 1st 2 wks of life
Adults then present with depressive behaviour
How is obesity and metabolic syndrome induced by diet?
- High fat diet fed to rats
- Cafeteria diet: regular chow + ad libitum access to energy dense foods e.g. cookies, cheese, processed meats etc.
Examples of surgical and physical alterations to create animal models of disease
- Ligating or cutting structures - e.g. LCA to generate MI
- Banding or constricting structures - aortic banding leads to cardiac overload, hypertrophy and eventually HF
- Removing organs - pancreatectomy to induce diabetes
Examples of genetic models of disease
- Forward vs reverse genetics
- Microinjection- induced transgenics
- Cre-lox system
Forward vs reverse genetics
Forward: generating spontaneous mutations - not ideal, you can’t target anything, unfair for the animals
Reverse: target specific genes, delete them, see the effects of that change on the phenotype
How does reverse genetics work?
- Inject stem cells into an egg grown by a donor mother
- Mutation incorporated into genome by homologous recombination
- Injected into a foster mother
- Offspring are chimeras - have original and GM genes
- Use selective breeding to get the line you want
How does microinjection-induced transgenics work?
- Obtain eggs
- Eggs fertilised
- Microinjection of manipulated DNA into pronucleus
- If prior to cell division, contributes to all cells of organism
- Fertilised egg then inserted into pseudo pregnant foster mothers
- Mated with vasectomised males - their body then preps for pregnancy
- Offspring are transgenic
Example of a transgenic mouse used in research
SOD1-G93A transgenic mouse model used in ALS (amyotrophic lateral sclerosis) research - display neurodegeneration and symptomatology consistent with ALS
the Cre-lox system
- Cre recombinanse gene is there
- LoxP sequences flank the DNA you want to delete (the gene is then called ‘floxed’)
- Add Cre recombinase, causes deletion of the floxed gene
When is the Cre-lox system used?
Can be used to generate general knockouts, but used more often to generate conditional mutations
What can be linked to Cre recombinase gene?
Promoter
Cre-lox can be either
Conditional on time or tissue-specific
Example of a knockout generated by the Cre-lox system
Cardiomyocyte-specific STAT-3 knockout - whole body STAT-3 k/o results in rapid degeneration of STAT-3 k/o embryos between days 6.5 and 7.5 in mice
How to get heterozygote STAT-3 k/o mouse
Cross STAT-3 floxed mouse (STAT-3 gene with LoxP either side) with a mouse with normal STAT-3 genes and Cre recombinase genes with heart-specific promoter
How to get a STAT-3 complete k/o
Cross STAT-3 floxed mouse and Cre-recom mouse
What is a mouse model of hyperlipidaemia and what can it be used to study?
Apolipoprotein E knockout mouse model, used to study atherosclerosis
What are usually used when generating new genetically altered mouse strains?
Usually wild type inbred strains or F1 crosses between two strains are used
What are outbred stocks?
Outbred colony: stocks
- A breeding group of genetically heterogenous animals
- Maintained in a close colony without the introduction of animals from another stock or strain
- Degree of genetic variation depends on the history of the colony
- Outbred stocks available for all species of lab animal
What is an inbred strain?
Inbred colony: strain
A strain which has been derived from 20 or more generations of brother-sister mating (and is therefore homozygous throughout almost all of its genome)
Advantages of outbred stocks
- cheaper to buy or breed
- more prolific
- can breed on a large scale with relatively little waste
- more readily available in groups of a defined weight or age range
Disadvantages of outbred stocks
- each animal is genetically unique
- no info. on the genotypes of individuals unless each is specifically genotyped
- individuals differ because of both genetic & non-genetic factors
- larger no. of animals needed to achieve this given level of statistical precision
- stocks are subject to genetic change as a result of inbreeding and/or directional selection, leading to changes in gene frequency
Properties of inbred strains
- Isogenicity: virtually genetically identical
- Decreased variation in results
- Clear genetic profile makes quality control easier
- Increased statistical precision - Homozygosity: 98% of same alleles at given locus
- Phenotypic uniformity: only variation should be due to non-genetic causes
- Long-term stability: no genetic variation is present so directional selection will be ineffective in changing geno/phenotype
- Individuality: each strain is genetically unique & has unique phenotypic characteristics
- more or less likely to get certain kinds of tumours - Sensitivity: more sensitive to environmental influences than outbred stocks/ F1 hybrids
◆ Increases need to make sure environment is kept constant even further
◆ May be more sensitive to experimental treatments
6 properties of outbred strains
- Isogenicity
- Homozygosity
- Phnenotypic uniformity
- Long-term stability
- Individuality
- Sensitivity
Advantages of inbred strains
- Clear genetic profile makes quality control easier
- Increased statistical precision
- No genetic variation is present so directional selection will be ineffective in changing geno/phenotype
Disadvantages of inbred strains
- Decreased variation
- More sensitive to environmental influences than outbred stocks/F1 hybrids
What are F1 hybrids?
- First generation cross between two inbred strains
- The offspring will be heterozygous at all loci where parental strains differ
- Useful themselves but will not breed true
Advantages of F1 hybrids over inbred strains
- More vigorous
- Less sensitive to adverse environmental conditions
- Live longer
- More robust than inbred strains
- Being isogenic, they have many of the more useful properties of the parental inbred strain
Use of F1 hybrids in research
- More robust than inbred strains
- Useful as foster mothers for production of transgenic strains
- E.g. NZBNZWF1 used as model of autoimmune SLE
Standardised genetic nomenclature for mice and rats
- Standard strain names in labs and codes to designate sub strains
○ C57BL-strain, sub strain 6J = line number 6 from the Jackson lab - Standard abbreviations exist for some strains
○ i.e. C57Bl/6J known as B6
