L-25 Flashcards
How can we get information about the function of a gene using its phenotype?
- by studying organisms that are naturally mutant for a particular gene
- increase the rate of random mutation, select for a phenotype of interest and sequence the genome to identify the mutation (genetic screen)
- take a gene you are interested in, copy it and insert it into another organism (transgenesis/ gene knockout/ reverse genetics)
What was the first example of a sex linked mutation in an animal
White eye phenotype in fruit flies
What is the human version of the white eye fruit fly gene mutation and what is it responsible for?
ABCG1 gene makes a protein required for cholesterol and lipid transport into cells
What is myotonia congenita?
An inherited condition caused by a defect in the CLCN1 gene which encodes a chloride channel receptor CLC-1
What are the symptoms of myotonia congenita?
Failure of muscles to relax after contraction, due to impairment in Cl- ion transport
What are the best model organisms to investigate gene function in humans?
- mice
- zebra fish
- drosophila
What percentage of our genome do we share with mice?
92%
What percentage of our genome do we share with zebra fish?
70%
what percentage of our genome do we share with drosophila?
44%
How do we carry out a genetic screen?
Treatment of model organism gametes with mutagens such as X rays or chemicals . Mutations made this way are random and can give quite dramatic phenotypes
What is transgenesis?
Engineering a multicellular organism by adding in “foreign” DNA.
What is the use of transgenic DNA?
To determine how certain genes work and what they do
- to engineer recombinant proteins
- gene therapy approaches to genetic disease
What does CRISPR stand for?
Clustered regularly interspaced short palindromic repeats
What does Cas9 stand for?
CRISPR associated protein 9
Where did Cas9 evolve and what for?
Evolved in bacteria for antiviral defense
What are the steps to making and using CRISPR Cas9:
- design a short “guide” RNA that only binds to your gene of interest
- Combine it with CRISPR protein to create a Cas9-guide RNA complex
- Insert this into the cells of interest in your model organism (not simple)
- Cas9 enters nucleus and finds target sequence in genome that matches guide RNA
- Cas9 makes a double stranded break in DNA at the target site
- in the absence of a template , DNA repair enzymes try to patch up the cut
- This often results in errors and InDels are created at the target site, the gene is potentially disrupted or mutated
- if a repair template is provided, it is possible to use this to “edit” the DNA sequence at the cut site
- this is called gene editing
What is a gene therapy treatment for cystic fibrosis?
Delivering DNA with a functional copy of the CTFR gene to the epithelial cells of the lungs via nebuliser. This extra copy makes good CTFR protein which restores function to some cells
What is an example of gene editing with CRISPR? Explain:
Foetus’s with sickle cell disease make haemoglobin differently, using alpha and gamma chains (HbF). A gene called BCL11A shuts down the gamma genes at birth. CRISPR-Cas9 was used to break BCL11A in the patients extracted bone marrow cells. The modified bone marrow cells were returned to the patient. Red blood cells now make HbF
Explain pre-implantation genetic diagnosis:
In families with an identified risk, IVF can be used to make embryos from the parents eggs and sperm. These embryos can be tested before implantation, and only healthy embryos will be implanted
Explain 3 parent babies:
When the faulty gene is on the mitochondrial DNA, nuclear transfer to a donor egg can be used
