Large scale genetic screens in zebrafish III Flashcards
What is injected into a fish alongside the tol2 mRNA (transposase)
The trap casette
What is the outcome of injecting the Tol2 mRNA and the trap casette?
Many trap copies are inserted into the genome of the fish, some of which go through the germline
What is done with the fish that have the trap cassette inserted into their genomes?
They are crossed w/ a WT fish
What is the result of crossing the trap cassette fish with a wild type of fish?
A progeny, each individual fish having its own mutation–> different to each other
What is done with the progeny of the cross of the WT and trap cassette fish?
Shine blue light on them to see where the GF is–> will be able to see what is mutated
What do you do once you see a fish with a mutation that you want to study?
Mate two of them–> 1/4 of the progeny will be homozygous for the mutation
First step of finding the gene that is responsible for the GFP?
Isolate the DNA and digest it w/ a restriction endonuclease
What is done after isolating and digesting the DNA from the fish?
The DNA is circularised by adding a ligase
What is done with the circularised DNA?
PCR with primers that bind to sequences in the trap cassette–> only circular DNA containing the sequence will be amplified
What is done with the PCR amplified sequences?
Sequenced–> compared to a database so you can ID the gene
What is reverse genetics?
Investigations on a known gene, especially by creation of loss-of-function mutants and/or transgenesis to overexpress the gene of interest.
How does reverse genetics work?
Start with the gene you want to ID the function of, mutate it, and look for changes in the phenotype
Issues w/ reverse genetic screens?
Cumbersome and expensive to do on a large scale
What is transgenesis?
Introduce a novel gene into the genome
What is targeted mutagenesis?
Gene knock out or knock down
Effect of morpholino oligonucleotides and RNA interference?
Mess up RNA function
What is transgenesis?
the process of introducing a foreign DNA into a living organism through random integration into the genome with the aim to stably express it
How are transgenics created?
microinjections of DNA plasmids into one-cell zebrafish embryos
How can spatial expression of a gene be controlled?
GAL4 UAS system
How does the GAL4 UAS system work?
GAL4 is a protein that binds to an upstream UAS sequence
Take a tissue specific promoter and put it upstream of GAL4, GAL4 is driven in that tissue
Also have a responder gene w/ a UAS sequence upstream of the gene of interest
Where GAL4 is expressed, your gene is expressed
How can temporal expression of a gene of interest be controlled?
Use a heat shock promoter–> heat up the fish when you want the gene to be expressed
How do most people mutate a gene of interest?
CRISPR-Cas9
