Drosophila Flashcards
Briefly describe how the UAS/GAL4 system works and how you can use the USA/GAL4 system to inhibit the gene NLaz specifically in neurons.
The UAS/GAL4 system is a binary expression system. It means that it can allow investigation of gene function through expression manipulation in targeted cells or tissues(knock down gene expression).
It is made from 2 major compartments: The GAL4 gene(derived from yeast genome) which encodes for the GAL4 transcriptional activator protein. The gene is put into the flies under the control of a native(only active in certain tissues) enhancer. When GAL4 protein is bound to UAS(upstream activation sequence), transcription of a reporter gene(gene of interest) will occur.
Mate flies – one with a driver line and one with a reporter line. You will get a generation of flies that contains both GAL4 gene and reporter gene. The enhancer connected to the GAL4 gene will enable transcription only in specific types of cells in the fly, due to being native/tissue specific. So the expression of the gene of interest can be controlled thanks to that enhancer. Depending on which enhancer we choose, we can choose where we want the gene of interest to be expressed.
In this case, we want to choose an enhancer specific for neurons. This time we want to express a gene whose product inhibits the NLaz gene. This will result in the GAL4 being expressed in neurons only, and the product from our gene of interest will inhibit the NLaz gene.
You have received a fly line from “the Bloomington drosophila stock center”. This fly line has a mutation in the 2nd chromosome that is balanced with cutly(cu). What is a balancer? What are the properties of balancers any why are balancers used?
Balancer chromosomes are genetic tools used to prevent genetic recombination between homologous chromosomes during meiosis. They are often used to maintain a population of flies through generations.
Balancer chromosomes have three main characteristics:
Multiple nested inversions -> prevents the chromosome from undergoing homologous recombination during meiosis. The mutation will stay on its chromosome.
Dominant markers/phenotypes -> easy pick out flies with certain phenotypes without genetic analysis (useful markers for this are curly on chr. 2, stubble on chr. 3)
Carries alleles that are lethal when carried homozygously -> any offspring obtaining both balancer chromosomes will die.
Usage of this results in several things. When crossing heterozygous flies there are three possible genotypes:
1. Offspring with homozygous mutations will not have a distinct phenotype(look WT).
2. Offspring with homozygous balancer chromosomes will die.
3. Heterozygous offspring will live. The mutation will stay on the sister chromatid because of the balancer chromosome traits.
In this case, since the homozygous mutated offspring is sterile, the only genotype who will live is the one with a single balancer chromosome.
Drosophila researchers make great use of forward genetics screens. To induce the mutations, researchers can use either chemical mutagens or transposons. Compare the advantages and disadvantages with these two methods
Chemical mutagen:
Hits the genome randomly
Generate different types of mutations: (lof, gof, temp sensitive alleles, etc)
-Tedious to identify the mutated gene.
Transposon-mediated:
Easy to identify the insertion site
Can detect expression pattern and protein localization
-Insertion is not completely random.
Drosophila researchers make great use of transposons as genetic tools. These can be designed in different ways, so that they can be used as mutagens, enhancer traps, gene traps or protein traps. Explain how transposons are used as gene traps.
Gene traps have a splice-acceptor site (SA) but no splice donor site. Transosase will insert the transposon almost randomly. IF it is inserted in an intron, any exons beyond the point of insteration will be excluded from the resulting fusion protein. By using a reporter gene (e.g GFP) as transposon we can do ransom knock-out while tracing protein location.
Imagine that you are a Drosophila researcher: You would like to knock- down the expression of a specific gene in the development heart, using RNA interference (RNAi). How would you achieve this? (explain the cross you would have to set up and how the method works).
Kommer på tenta
I order flies that express Gal4 only in the heart.
I then create the following construct UAS, inverse repeat,
This construct is used to make transgenic flies (not the same flies). The inverse repeat is conjugate to the gene mRNA. The Gal4- flies and these flies are cross-bred, which causes Gal4/UAS- offspring that produces hairpin- RNA in the heart. The dice- system digests hairpin-RNA into ds-RNA which knocks down the gene.
What are “polytene chromosome” in Drosophila?
Polytene chromosome are a strongly amplified form of interphase chromosome, found in ex salivary gland cells of Drosophila.
You are joining a lab to study Alzheimer disease in Drosophila. For that you will express the human amyloid beta42 peptide in flies. What are the advantages/disadvantages of using drosophila as model organism in this investigation? What kind of experiments would you propose to evaluate the toxicity of the Abeta42 peptide in drosophila?
Advantages with flies: Easy and cheap to grow. Genetic manipulations are relatively easy. Short life span which allows for multi generation experiments.
Disadvantages with flies: They don’t work exactly as humans do; results may not be directly translated. Short lifespan could also be bad when studying things that have long term effects that are of relevance, altzheimer being such etc.
We could use behavioural assays. With these we can measure/analyse several things.
Food -> are their eating patterns deviating? Measure food intake with dyed food and spectrophotometer
Sleeping -> are they sleeping more or less? Does the treatment affect the brain regarding sleep cycle?
Climbing -> how is their mobility and coordination affected?
Mating -> are their courtship behaviour affected?
In the context of Drosophila genetics, describe an “enhancer trap”. Your answer should explain what enhancer traps look like structurally, and what they are useful for.
An enhancer trap is used to express a marker gene with help of the enhancer of another gene.We create a transposon with a reporter gene, GFP etc. The transposon is silent in absence of enhancers, since the marker gene only has a minimal promoter. When it is integrated into another gene(in the introns) the target gene and the GFP will have the same expression pattern. If the transposon is integrated into an exon, the protein will be disrupted/non-functional. This can be used in forward genetics to find out which gene the transposon was integrated in.
Karl is one of your favorite Drosophila proteins. You would like to produce this protein in large amounts to do some in vitro experiments. How do you pursue it?
We can use the Drosophila S2 cell line and cultivate them in vitro(immortal cells). A large yield is possible in these cultures. It is very suitable since they are from the same organism and have the same genotype, so the proteins are an exact match with all the post-translational effects etc.
Your lab is about to start a forward genetics screen (in flies) using transposons. You would like to get null mutations, so you plan to do a gene trap screen. However a lab mate insists you should do a Protein trap screen. Which system (Gene or protein trap) has a higher chance to give null mutations? Why?
Null mutation= when a gene usually transcribes lets say an enzyme, but when mutated transcribes nothing or a defect protein.
The finished protein trap product when spliced, will have a reporter gene in it, between the exons, in the introns. So the protein will have, lets say GFP, within it. You cannot know for sure if this will disrupt the protein, sometimes it does and sometimes it doesn’t. Since all the exons are still intact you cannot 100% say that the protein wont fold correctly-> have its function still intact.
However, with gene trap, you insert the reporter gene with a STOP codon in it, at the 3’ end. This means, that even when the product is spliced and all, the product(protein) will never be functional, since some exons are not present. (The reporter gene will be expressed, so it will be visible under a fluorescent microscope etc., so you know that the process of transcribing worked).
You have just received a fly in line from “The Bloomington Drosophila Stock Center”. This particular fly stock has a null allele of the AdipoR gene (no AdipoR protein is produced)
The AdipoR mutation is balanced with TM6C, Sb in the 3rd chromosome. Why do we need to balance some fly stocks? As always, one should be careful when new flies arrive at the lab. What is the main external threat for fly stocks? Now, you plan to do transgenesis and reintroduce AdipoR expression in specific tissues using the UAS/GAL4 system. But first how does the UAS/GAL4 system work?
We need to balance some fly stocks because they have a mutation that means something to us, and we want to “keep it alive” so we can study it whenever we want to.
The main external threat for Drosophila are contaminations, in form of other organisms that disrupt their living. Mite, bacteria, and fungi are common. These can be treated with antibiotics or living yeast. Wolbachia bacteria lives within the flies and impact their phenotype. The flies can be treated with tetracycline to treat Wolbachia.
