model organisms Flashcards
define what a model organism is
an organism chosen for the study of another organism, for any or all of the following reasons: small genome size, short generation time, ease of manipulation in genetic experiments
what are 8 features sort after in a model organism?
Small genome size
Generation time
Genome sequenced (tho this is easy enough, but if it has already been sequenced its likely some work has been done that you could look at)
Easy to grow
Abundant progeny (especially if trying to detect rare events)
Can self-fertilise OR cross - you can get an inbred line with zero genetic variation if so desired, but sometimes you want to combine different genomes so both abilities is ideal
Tools for Genetic Modification - so you can alter genes
Mutant collections and genomic resources - easiest way to identify what a gene does is ordering a mutant of it, knocking it out
mice can be crossed, but cannot self-fertilise. how do geneticists get around this?
two other shortcomings?
they are inbred instead
their genome isn’t that small
their generation time is on the longer side
mice - what are some logistics about life cycle/genome etc… important to their usefulness?
mus musculus - a mammal
99% of mouse genes have human homologues - have a human gene doing the same job in the same way (conservation of sequence and function)
Diploid life cycle, XY sex-determining system
Life cycle = 10 weeks, a female can have 5 litters of 5-10 pups
Great for genetic basis of cancer, carcinogen toxicity, behavioural genetics, developmental genetics and immunology
Typical tools - transgenesis (addition of a new gene) and targeted gene knockouts
what is meant by homologues?
have an e.g. human gene doing the same job in the same way
conservation of sequence and function
choosing a method to produce a model for human disease depends on…?
two routes?
the inheritance pattern of the disease
recessive - like CF, you must knock out the healthy copies of the gene, so this is targeted gene example, or gene replacement
dominant - like spinocerebellar ataxia 1 - only need one copy of the dominant gene, so this is an ectopic insertion
explain the steps involved in gene targeting in mice up to getting your embryonic stem cell that has successfully taken up your target vector
Get some mouse embryos that have not implanted, and remove some embryonic stem cells from the inner cell mass (the outer cells are for forming the placenta). Cultivate these cells
Make your targeting vector -
This should be DNA homologous to your target gene - e.g. cystic fibrosis gene (but the mutant you desire) - with a positive tag sandwiched in the middle so you can identify cells which have taken up the target gene. There is also a negative tag on the end to kill cells that only manage to take up this part?
Transfection - cells may perform homologous recombination, allowing your target vector to find and be integrated into the target gene
Positive-negative selection - the correct homologous recombination occurring relies on chance, so you would screen for your positive tag (and kill cells with the negative tag - e.g. apply a chemical that kills cells with the HSV-tk gene thingy seen in the diagram)
You would isolate and grow this ES cell that has successfully taken up your target vector
once you’ve got your embryonic stem cell that has successfully taken up your target vector, what’s the next step? (mice)
- inject the ES cells into blastocysts where they mix and form a mosaic with the cells of the inner mass, from which the embryo develops (i.e. some with the target gene, some with WT)
- implant the blastocysts into a surrogate mother, where they develop into mosaic embryos
- the mice that are born are mosaic
- not all the mosaic mice will have the mutant we inserted in their germline cells, you mate them with normal mice to produce both hence mutant and normal offspring. success (the mutant offspring)
- An additional step may be required, e.g. inbreeding these knockout mice to get specimens with two of the inactivated target gene (homozygous)
what is cystic fibrosis?
Single-gene disease characterised by the buildup of thick, sticky mucus that can damage many of the body’s organs.
Results in progressive damage to the respiratory system and chronic digestive system
Mutations in the Cftr gene (Cystic Fibrosis Transmembrane conductance Regulator), affecting transmembrane chlorine ion transport across the membrane of epithelial cells
1 in 3000 white individuals (less common in other ethnic groups)
how were cystic fibrosis mice made?
CF (most commonly) is a 3 base pair deletion (delta for deletion, F for phenylalanine, 508 for the position of this Aa) called ΔF508 protein / so made the ΔF508 mouse.
- Produced linear DNA via PCR
- 3/124 ESCs correctly underwent homologous recombination upon transfection via electroporation
- Injected the embryonic stem cells into blastocyst to produce chimeric mice
- Bred with WT mice to give heterozygous mice ((ΔF05+/-)
- Inbred to give homozygous mice
Once symptoms were confirmed, was used as a model to test gene therapy
what is spinocerebellar ataxia?
cerebellar degenerative disorder
Initially experience problems with coordination and balance (ataxia)
speech and swallowing difficulties
muscle stiffness (spasticity) and cognitive impairment, sensory neuropathy, dystonia and atrophy
tend to survive 10-20 years after symptoms first appear
what causes SCA1?
Broadly, what was done in terms of making a SCA1 mouse/making the transgene?
its caused by enlarged CAG repeats (polyQ) in the ATXN1 gene (39-83 repeats are seen in the disease)
Made a SCA1 mouse with an allele with 30 repeats as a control, and then with 82 CAG repeats to mimic the disease
Chose a promoter murine Pcp2, supposed to be capable of directing transgene expression specifically to purkinje cells (neurons involved in ataxia)
They checked their transgene was on with the promoter they used - found 10-100 fold greater levels of expression (paper said this was good, but we are sceptical, surely you would want expression to be normal)
the SCA1 mouse was made by pronuclear injection. how does this work?
Take a fertilised egg, before the egg and the sperm pronuclei have fused
Inject DNA into the male pronucleus
Nuclei fuse as normal
Implant into the mother, look at the resultant mice (founder gen, F0)
They are either
- Transgenic with DNA being integrated into sperm pronucleus, before the pronuclei fuse and the diploid divides. They are hemizygous as the transgene only comes from dad (sperm pronucleus?)
- WT with the injected DNA not being integrated
- Mosaic, with the transgene only being integrated after nuclear fusion and a few divisions have already occurred
To produce a stable transgenic lineage you must do the inbreeding stage of F0, until you have a pure breeding line
what did analysis of the transgene expression show?
Performed tail biopsies (took end of tail and analysed some DNA) used PCR and southern blot
There was no homologous recombination, it was ‘nonhomologous’, i.e. occurs anywhere in the genome (could interrupt an important gene for example and therefore is not ideal)
also more than one copy could be integrated and not necessarily together - there was no quality control
Used a GFP tag to see which mice have the transgene and are expressing it
what discoveries were made from the SCA1 mouse?
(in mice with the 82-repeats transgene of ataxin-1)
Short strides at 12 weeks, gait issues at 1 year, and diminished performance on the rotarod test obvious by 5 weeks (can’t balance on a rotating rod for as long)
Impaired degradation of ataxin-1 by the proteasome is linked to pathogenesis in SCA1 mice (accumulation of the protein, like in Huntington’s)
Severity of the phenotype is linked to nuclear localisation (aggregation) of the ataxin-1 protein (worsened symptoms if the protein aggregates are in the nucleus)
