Investigating the Function of Individual Genes Flashcards
How do we get information about the function of a gene from its phenotype?
- By studying organisms that are naturally mutant for a particular gene, we can work out what that gene might do
- Where no natural mutant exist we can make our own
- By studying both these types of mutants we can learn how particular mutations lead to phenotypic changes
Natural Mutants - Describe how where the genetic change alters the phenotype, gives clues to gene function
- The phenotype is what we see. In this case it is polydactyly (two middle fingers)
- The cause of this phenotype is a mutation in a gene
- The normal role of this gene is to prevent this phenotype
- What is the normal function of this gene? The patterning of the hand
What is the value of mutants?
- While variation in the human genome is common, most of this does not affect the phenotype
- Mutations are rare, they are a subset of variation, but do not always affect “fitness”
- Around 20% of genes have an unknown function
- Luckily, many of these genes are conserved in other animals - even, in many cases with fruit flies and yeast. If we can find or create mutants in these related genes, we may learn their functions in humans
How do we use genetic techniques to find out what a gene does?
- Study organisms that are naturally mutant for that gene (rare!)
- 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/genetic engineering)
- Deliberately break a particular gene to see what happens (targeted mutation/gene knockout/reverse genetics)
- This type of approach is called functional molecular genetics
Describe how model organisms can be used to make mutants
- We share many of our genes with other animals
- Model organisms are ones that can be easily raised in a controlled environment and are easy to manipulate genetically
- Each organisms has a different approach that works best for making changes to the DNA genome
Eg. fruit flies, mice, zebrafish
What is transgenesis?
Taking a gene you are interested in, copying it and inserting it into another organism
- The DNA code is universal, so any DNA can be used by any organism - even synthetic DNA
- Engineering a multicellular organism by adding in ‘foreign’ DNA is knows as transgenesis
- We can use transgenic DNA to understand how genes work, to engineer recombinant proteins (synthetic biology), or in gene therapy approaches.
Requires a regulatory sequence (that says pls turn me into a protein) attached to the gene.
How do we know if a gene variant is pathogenic?
Modern genetics targets mutations to the DNA sequence of your choice to ‘break’ specific genes
- We can damage, or modify, the gene we are interested in by genetically modifying an organism or cell line
- By examining the organism, or its offspring, we should be able to work our what the gene normally does.
- There are many ways to do this, but we will look at one: CRISPR - Cas9
Describe targeted mutation with CRISPR-Cas9
- CRISPR = clustered regularly interspaced short palindromic repeats
- Cas9=CRISPR associated protein 9
- Evolved in bacteria for antiviral defense
- Decide which gene you wish to mutate
- Design a short ‘guide’ RNA that only binds to your gene of interest
- Mix these together
How does CRISPR-Cas9 get into the cells of interest?
- Cas9 enters the nucleus and finds the target sequence in the genome that matches the guide RNA
- Cas9 makes double stranded break in DNA at target site
- In the absence of a template, DNA repair enzymes try to patch up the cut.
- This often results in errors as there is no template to read from
- Small InDels are created at the target site, the gene is potentially disrupted, or mutated
- If repair template is provided, it is possible to use this to ‘edit’ the DNA sequence at the cut site - ‘gene editing’
Genetic disease: can we fix it? (somatic)
Yes we can (only if we know what causes it and have a way to correct the defect.
Somatic: (trying to fix the sick person, not editing the human race)
- Target the cells or organs effected
- Does not affect the next generation (is not a change in the gremline)
- Gene therapy example: Cystic fibrosis; one of the most common lethal single gene genetic conditions, defect in CFTR gene, which codes for a chloride ion transporter
- Gene editing with CRISPR-Cas9 example: Sickle cell disease; mutation in haemoglobin, the oxygen carrying protein in red blood cells
*Note: the CRISPR edited babies allegedly created in 2018 by He Jianguo are germline mutations
Describe how gene therapy for Cystic Fibrosis works
Delivering DNA with functional copy of CFTR gene to lung epithelial cells via nebuliser.
Extra copy makes good CFTR proteins, restoring function to some cells.
In easier to understand terms we are adding a small bacterial plasmid by wrapping it in fake plasma membrane. It then fuses with the plasma membrane so the plasmid is released into the lung cells and into the nucleus. It then makes copies of the genes.
Describe how fixing genetic disease in Germline cells works
We can do it, but only of we know what causes it, have a way to correct the defect, AND have considered ethics.
Germline:
- 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 implanted.
- Three parent babies: where the faulty gene is on the mitochondrial DNA, nuclear transfer to a donor egg can be used. (Although, there are risks with this because to transfer a nucleus you have to take some mitochondria with it. Sometimes this can result in these mitochondria outcompeting the good mitochondria so these kids are getting symptoms)
- CRISPR gene “edited” babies: (Jiankeui He, unpublished)
