Lecture 25 Flashcards
How do we get information about the function of a gene from its phenotype?
- by studying organism as that are naturally mutant for a particular disease, we can work out what that gene might do
- where no natural mutants exist we can make our own
- by studying both these types of mutants we can learn how particular mutations lead to phenotypic changes
What causes a change in phenotype
- the cause of phenotype change is a mutation in a gene
- the normal role of the gene would be to prevent this phenotype
- what is the normal function of this gene
The effect and value of mutants
- although variation in the human genome is common, most of the is down not affect phenotype
- mutations are rare, they are a subset of variation, but do not always effect “fitness”
- around 4000 (20%) human genes have unknown function
- many of the unknown genes are conserves in other animals, meaning if you can find or create mutants in the related genes, you can learn the functions in humans
Thus, castigation is common but phenotype-causing mutations are rare
How do you use genetic techniques to find out what a gene does: functional molecular genetics
- study organisms that are naturally mutant for that gene
- increase the rate of random mutation, select for a phenotype of interest and sequence the genome to identity the mutation (genetic screening)
- take a gene you are interested in, copy it and insert it into another organism (transgenesis/genetic engerneering)
- deliberately break a particular gene to see what happens (targeted mutation/gene knockout/ reverse genetics)
Model organisms can be used to ____ ______
Make mutants
What are model organisms
Model organisms are ones that can be easily raised in a controlled environment and are easy to manipulate genetically
- these can be used to make mutants as we share many of our genes with other animals but each organism has a different approach that works best for making changed to the DNA genome
Transgenisis
- when you move DNA around to understand hoe the genes work
- 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 known as transgenesis
- we can use transgenic DNA to understand how genes work, to engineer recombinant proteins (synthetic biology), to in gene therapy approaches
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 modifiying an organism or cell line
- by examining the organism, or its offspring we should be able to work out what the gene normally does
- many ways to do this but we will look at one: CRISPR-cas9
What is CRISPR? And what is Cas9?
CRISPR = clustered regularly interspaced short palindromic repeats
Cas9 = CRISPR associated protein 9
How does tartgeted mutation with CRISPR-Cas9 work?
- evolved in bacteria for antiviral defence
- decide which gene you wish to mutate
- design a short “guide” RNA that only binds to your gene of interest - join them
- get this into the cells of Internet in your model organism (hard to do)
- Cas9 enters nucleus and finds target sequence in geno,e that matches guide RNA
- Cas9 makes double stranded break in DNA at target site
- in 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 (scientists can disable the target gene to study its normal function)
- if repair template is provided, it is possible to use this to “edit” the DNA sequence at the cut site - “gene editing” (if the target gene has a mutation, it can be repaired)
How to fix genetic disease - somatic
(Can only be done if we knows what causes it and have a way to correct the defect)
Somatic:
- targets the cells or organs effected
- doesn’t effect next generation
- gene therapy example: Cystic fibrosis, one of the most common lethal single gene genetic conditions, defect in CFTR gene, which codes for a chlorine ion transporter
- gene editing with CRISPR-Cas9 example: sickle cell disease, mutation is haemoglobin, the oxygen carrying protein in red blood cells
Gene therapy (cystic fibrosis)
- delivering DNA with functional copy of CFTR gene to lung epithelial cells via nebuliser
- extra copy makes good CFTR protein, resorting function to come cells
Genetic disease, can we fix it? GERMLINE
(Only if we know what causes it, have a way to correct the defect and have considered ethics)
Pre-implantation genetic diagnosis:
- in families with an identified risk, IVF can be used to make embryos from he parents eggs and sperm. These embryos can be tested before implantation and only health embryos implanted.
Three parent babies:
- where the faulty gene is on the mitochondrial DNA, nuclear transfer to a donor egg can be used
CRISPR gene “edited” babies
