Lecture 8 - Mouse as a model for Human Disease Flashcards
(30 cards)
(self-assessment question) Which disease is mostly caused by genetic factors?
(Figure given)
- biggest difference between fraternal twins & monozygotic twins in Bipolar disorder
(self-assessment question) Why are inbred lines useful in studying quantitative traits?
There is no genetic variation so all observed variation has to be due to the environment
(self-assessment question) Monozygotic twins become increasingly different from each other during their lifetime. What is causing this change?
Increasing portion of their life is influenced by environmental factors. Different epigenetic (methylation) markers accumulate over a lifetime
What are examples of model organisms?
- zebrafish
- C. elegans
- Yeast
- Drosophila
- Arabidopsis
- Mice
What is a model organism?
An organism that is chosen for the study of other organisms because of any or all of the following factors:
- small genome size
- short generation time
- ease of manipulation in genetic experiment
What are common features of model organisms?
- small genome size (compared to actual study organism)
- generation time
- easy to grow
- abundant progeny
- can self-fertilize OR cross (not all e.g. mice)
- tools for genetic modification
- mutant collections & genomic resources
Describe mice as mammals
- Mus musculus
- 99% mouse genes have human homologues
- Diploid life cycle with XY sex-determining system
- Life cycle: 10 weeks. A typical female can have 5 liters of 5-10 pups a year
- Useful for: genetic basis of cancer (and carcinogens), behavioural genetics, developmental genetics, immunology
- Genetic tools: transgenesis & targeted gene knockouts
What human diseases has a mouse model been used for studying?
- cancer
- diabetes
- cystic fibrosis
- hypertension
- cognitive-affective syndromes (an example of neurodegenerative diseases)
- cardiovascular diseases
- Thalassemia (haemoglobin)
- Developmental disorders
What models are used to study HIV?
- cats (FIV)
- monkeys (SIV)
What are transgenic mice?
- there is a lot of mouse-tech out there
- often the aim is to produce a model for human disease
- I have to chose one ‘targeted’ & one ‘ectopic insertion’ example:
Gene Replacement - Cystic Fibrosis (CFTR gene)
Ectopic insertions (transgenesis) - Spinocerebella ataxia (ATXN1 gene encodes the ataxin-1 protein) - insertion of gene but not deciding where the transgene goes
What is the general strategy for gene targeting in mice?
- ES cell culture
- Construction of targeting vector
- ES cell transfection
- Proliferation of targeted ES cell
- Injection of ES cells into blastocysts
- Birth & breeding of mosaic mice
- Production of homozygous knock-out mice
What occurs during ES (embryonic stem cell) cell culture?
ES cells are cultivated from mouse pre-implantation embryos (blastocysts)
Pre-implantation embryos & take cell mass from inside - outside becomes placenta & inside - outside becomes placenta and inside becomes baby mouse. ES cells taken from centre and cultured in-vivo
What occurs during Proliferation of targeted ES cell?
The vector contains pieces of DNA that are homologous to the target gene, as well as inserted DNA, which changes the target gene & allow for positive-negative selection.
Has 2 arms of homologous DNA & inside has a selectable marker. DNA repair using homology. Has a negative marker which is lethal - if it makes it inside genome of cell it will lead to death. This is to ensure both arms have undergone recombination. This is positive/negative selection.
What occurs during ES (embryonic stem) cell transfection?
The cellular machinery for homologous recombination allows the targeting vector enable that target vector to find and recombine with the target gene.
Introduction of linear DNA into cell & cellular machinery does the homologous recombination. Selects for rare events where homologous recombination occurs at both arms. If it occurs correctly,: genome - homologous arm - neomycin resistant gene (selectable marker) - homologous arm - genome.
What occurs during proliferation of targeted ES cells?
End up with a pure population of embryonic stem cells that carry the target gene
What occurs during the injection of ES cells into blastocysts?
The targeted ES cells are injected into blastocysts.
The blastocysts are then inserted into surrogate mother (pseudo-pregnant) & first generation are mosaic mice. These mice have some cells with gene-targeted and some cells won’t
What occurs during birth & breeding of mosaic mice?
Breed mosaic mouse with normal mouse - using this, you can work out whether the cells that make sperm have had gene target or not.
What occurs during the production of homozygous knock-out mice?
This involves inter-breeding of gene-targeted mice.
Describe Cystic Fibrosis
- Single-gene disease characterized by the buildup of thick, sticky mucus that can damage the body’s organs.
- Results in progressive damage to the respiratory system & chronic digestive system
- Mutations in the CFTR gene (Cystic Fibrosis Transmembrane conductance Regulator) found on chromosome 7
- Affect transmembrane chlorine ion transport across the membrane of epithelial cells
- Occurs 1 in 3000 white individuals, less in other ethnic groups
How is a mouse used as a model for Cystic Fibrosis?
- a mutation was made by a 3bp deletion in the CFTR gene between 1522-1524 in exon10.
- This was called DeltaF508 (delta = triangle)
- Linear DNA (a PCR product) was transfected into TG4 embryonic stem cells by electroporation.
- 3/124 clones has correctly undergone homologous recombination
- Germline chimeric mice were generated by injected the cells into blastocysts
- Heterozygous offspring were inter-mated to produce homozygous lines
- Verified that the DeltaF08 mouse had CF-like symptoms
- Used to test disease models & gene therapy
- Example of gene targeting
What is SCA1: Spinocerebellar ataxia type 1?
- SCA1 is an autosomal dominant hereditary ataxia
- Caused by enlarged CAG repeats in ATXN1 gene
- Results in poly-glutamine tract (polyQ) extension of the ataxin-1 protein
- 6-25 glutamine repeats is normal
- SCA1 patients have 39-83 repeats
How was mice used as models in Cerebellar degenerative disorder (dominant)?
Case Study 2: Spinocerebellar ataxia type (SCA1)
- affects 1-2 in 100,000
- symptoms start with initially experience problems with coordination and balance (ataxia):
- speech & swallowing difficulties
- muscle stiffness (spacticity)
- may have difficulty processing, learning & remembering information (cognitive impairment)
- over time can develop numbness, tingling, or pain in the arms & legs (sensory neuropathy); uncontrolled muscle tensing (dystonia); muscle wasting (atrophy)
- people with SCA1 typically survive 10-20 years after symptoms first appear
How were transgenic mice generated to test disease models & gene therapy of SCA1 (spinocerebellar ataxia type 1)
- The transgene inserts were isolated by Sal-1 digestion, gel purified, extracted with organic solvent & extensively dialyzed prior to microinjection.
- Transgenic SCA1 mice carrying the allele with 82 CAG repeats
- The murine Pcp2 promoter, which is supposed to be capable of directing transgene expression specifically to Purkinje cells
- 5 lines of the mice showed transgene expression levels 10-100 fold greater than levels of endogenous mouse ataxin-1 mRNA
What is the process of generating transgenics via pronuclear microinjection into fertilized eggs?
Mice will be hemizygous (one gene) & may be mosaic - production of a stable transgenic lineage requires a second inbred generation
Take fertilized egg before haploid pronuclei fused. Pipette to inject linear DNA into the male pronucleus. Allow nuclear fusion - diploid zygote. Implantation into foster mother & then analyze pups (F0 or founder population).
In theory, this should impact all cells in mouse. However, mosaic mice created. Needed to check cells produced to see whether germline have got transgene in them. Mice will be hemizygous - only in dad copy. Homozygous lines require interbreeding of next generation
Example of adding a copy of gene.
