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when was RNAi as a mechanism first used?



what organism does RNAi not work in?



do people know why RNAi doesn't work in zebrafish?



how was RNAi first discovered?

sense RNA was injected but didn't work and antisense RNA didn't work very well either, but they tried double stranded RNA this worked! first done in the c.elegan


what is the mechanism of RNAi gene silencing?

double stranded RNA is injected into he organism or introduced in some way. You have a protein called dicer which chops the double stranded RNA into short interfering RNA (siRNA). Then you have a protein belonging to he argonauts or piwi proteins which form a RISC complex (RNA-induced silencing complex) which binds to the siRNA and converts them to a single strand which is used as a guide. If the match is really good then the RISC complex will "slice" the mRNA that is complimentary (degradation) or it will induce translational repression


what is the human RISC called?



how many sub units does the argonauts protein have?



what is the function of the RISC complex?

it stretches the guide RNA strand and the active site which has 2 Mg atoms needed for the slicing function. The other 3 subunits help to modify the structure


how does RNAi cause transcriptional silencing?

siRNA is formed from dicer and then it binds to RITs, which act in the nucleus, they make sure the single stranded RNA binds to nascent mRNA while it is being transcribed. the consequence of this is the RITS inducing histone methylation, DNA methylation and transcriptional repression via chromatin condensation


why did the RNAi system evolve?

it is a defence mechanism from dsRNA viruses


what type of viruses incorporate into the genome? and how does this link to the RNAi mechanism

retroviruses - their transposons are often transcribed from both sides and then forms dsRNA which is then targeted by the RNAI


who can RNAI be supplied to worms?

They can be fed with e.clo expressing dsRNA against the gene because they have a dsRNA transport channel called SID-1 which allows the RNAi to spread across the body of the worm. It can also be injected. into the gut.


how can genome wide screens be carried out in c.elegans?

can fill 96 well plates with c.eleganas and each well contains e.coli expressing a different RNAi probe almost all of the genes in the worm's genome. You then inspect either the P0 worms or the F1 worms of these- the RNAi is passed onto the offspring. You can use reporters to then look for affected phenotypes


how can you carry out RNAi in drosophila?

use DNA directed RNAi- create a DNA plasmids from which you transcribe an RNA (double stranded) which contains a spacer in between the two homology arms so that you get a hairpin loop of RNA. then this triggers the RNA response. To introduce the this construct into the fly. You can induce the expression of the RNAi using the GAL4/ UAS system. You can have gal4 downstream of a universal promoter or in a specific tissue. If you keep flies at a cold temperature, Gal4 will be less active that at 29 degrees- just by changing temp you can modulate the level f gal4 expression


can the gal4/uas system be used in other organisms other than flies?

yes- zebrafish


how can Gal4 is repressed?

- via temp- via a reposer called gal80. there is a temp sensitive version of gal 80- at 18 degrees it will bind to gal4 and there is no transcription, at 29 degrees, gal80 changes its conformation and can no longer bind- this can allow you to temporally control your genes expression. so you could cross a fly that expresses. you normally have GAL80 expressed ubiquitously, then Gal expressed in a tissue specific manner and then cross this fly with a fly that expresses the UAS construct.


what must gal4 have attached to it in order for it to work in the UAS system?

a DNA binding domain


what is the split gal 4 system? and when would it be used

you have two different enhancers- one expressing the gal4 DNA binding domain and the other expresses the gal4 activation domain. If you know that there are two genes that are expressed in different places in the organism but you want to see where the expression overlaps then you can use this system by using the enhancers for the two genes with the two protein subdomains. Then you have a UAS driving GFP expression


how can you use the split gal4 system to subtract cells that express both of 2 genes of interest?

you have enhancer-1 driving the expression of gal4, then you have enhancer-2 driving the expression of gal80. then you have a UAS driving GFP expression. so that the tissues which express both enhancer-1 and enhancer-1 will be turned off.


how can you turn on gene transcription using gal4 via drugs? why is this a good method?

have a gal4 that is drug inducible. SO you have a tissue specific transcription of gal4 but only fed the flies are put on foo containing this drug- then you can temporally and spatially control the expression of a gene in a way that isn't temperature sensitive. This is a goo method because you have a good control- one group on the food and others not on the food.


what must always be considered when you are using mechanisms such as the gal4 UAS system?

you need to make a control for every gene that you introduce to ensure there are no indirect artefacts


describe two examples of RNAi being used in flies to look at clocks .

1.they were looking at circadian rhythms. The used a GAl4 system so with a looper and RNAi gene sites. they knocked down the gene period which is needed for the clock to fun. They made two constructs targeting different regions of the per gene. One was against the Ct domain and the other against the PAS. They timeless- gal4 (a gene expressed in the same cells and is a clock gene). They then looked at the behavioural rhythms. These flies were rhythmic but they have a slow running clock. 2. They wanted to knock down pigment dispersing factor in neuronal subsets. only 16 of the clock neurons express pigment dispersing factor. They want to dissect which neurons are important for the circadian function of PDF (PDF loss of function: they become active in the afternoon earlier than wild type flies, flies do not become active before the lights come on, when you put the flies in darkness they lose their close.) so which of these neurons are important for these diff phenotypes. They used 5 different gal4 drive which are expressed in a diff subset of PDF expressing neurons (they used the gal4 driven by expression of each of the to drive the expression of UAS galactosidase expression as a marker to find out which ones each is expressed in). First they investigated how well the RNAi was working. SO they stained the nervous system with a PDF antibody to show tis expression. Then they did this in tim-gal4/UAS-pdfpRNAi (tim is subset promoter) and they saw it was knocking out PDF. (did verification for all drivers) Then they analysed the behaviour of these flies.


what does a RNAi produce?

a knock down not a knock out- you reduce expression depending on the quality of the RNAi construct.


what are the limitations of RNAi and what are the remedies for these limitations?

1. off-target effects: RNAi targets different of additional mRNAs with similar sequence 2. partial gene function normally remains 1. you can use different RNAi constructs, targeting different regions of the mRNA- if you get the same phenotype by knocking down two different regions of the same gene, you are sure it is due to the KD of that gene and not another gene. You can rescue the phenotype by creating RNAi resistant rescue. This will show that the phenotype was only caused by the knock down of specific gene 2. You can boost the expression of the dicer machinery by using a UAS construct to drive the expression of another copy (but you have to add another construct then). You can also add more than one transgene of the RNAi construct. or you can add a copy of gal4 driver. You could increase the number of UAS-repeats.


how can you rescue RNAi induced phenotypes?

introduce a transgene of the target gene that cannot be KD by the RNAi, then the phenotype must be due to the RNAi of that gene. One way of doing this is to use a gene from a sufficiently divergent different species that still have the same function but is different enough to not be targeted by the RNAi. Or you can use the degenerate genetic code and then make a different DNA for the same protein using different triplets.


how are transgenes normally made in flies?

by P-element- mediated germline transformation- injected vector with gene of interest and with p-element surrounding and then with transposes.


when generating a transgenic fly, what must you include in the construct and what is the egenral genetic background that should be used? (CHANGE ANSWER DEPENDING ON RALPH'S REPLY)

You should use a genetic background that has a known phenotype that can be rescued by thee expression of another allele- for example use a strain of white eyed flies. You then included W+ gene (encoding red eye) in the construct next to your gene that you want to express so that you know your transfection has been successful when the fly expresses red eyes. Therefore, you would include the regulatory elements of w+ gene.


how many genes for the VDRC cover and what percentage of the genome does this cover? what is a bonus of this library? (2)

12,671 genes vs 15000??, (91%) of all dros protein coding genes. 8267 genes more than one RNAi line available and they have the KK line which inserts into known regions


why are c.elegans better for genome wide screens than flies?

the RNAi libraries available for c.leegans are more complete than those for the flies


what can you order for fly RNAi libraries?

you can order libraries for flies which contain DNA fragments containing all genes that are flanked then with a T7 RNA polymerase primers which you can then use T7 RNA polymerase to transcribe dsRNAs and transfect either in cells or into a fly strain by inserting it into a construct