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Flashcards in deck_4985544 Deck (54)
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1
Q

when was RNAi as a mechanism first used?

A

2003

2
Q

what organism does RNAi not work in?

A

zebrafish

3
Q

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

A

no

4
Q

how was RNAi first discovered?

A

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

5
Q

what is the mechanism of RNAi gene silencing?

A

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

6
Q

what is the human RISC called?

A

argonaute

7
Q

how many sub units does the argonauts protein have?

A

4

8
Q

what is the function of the RISC complex?

A

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

9
Q

how does RNAi cause transcriptional silencing?

A

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

10
Q

why did the RNAi system evolve?

A

it is a defence mechanism from dsRNA viruses

11
Q

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

A

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

12
Q

who can RNAI be supplied to worms?

A

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.

13
Q

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

A

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

14
Q

how can you carry out RNAi in drosophila?

A

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

15
Q

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

A

yes- zebrafish

16
Q

how can Gal4 is repressed?

A
  • 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.
17
Q

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

A

a DNA binding domain

18
Q

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

A

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

19
Q

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

A

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.

20
Q

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

A

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.

21
Q

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

A

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

22
Q

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

A

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.

23
Q

what does a RNAi produce?

A

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

24
Q

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

A
  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.
25
Q

how can you rescue RNAi induced phenotypes?

A

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.

26
Q

how are transgenes normally made in flies?

A

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

27
Q

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)

A

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.

28
Q

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

A

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

29
Q

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

A

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

30
Q

what can you order for fly RNAi libraries?

A

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

31
Q

how do the libraries differ?

A

they vary in how the construct is introduced into the fly, one has p-elements which means that the RNAi is randomly located into the genome. There is a KK line which uses phi integrase which allows you to precisely insert theh transgene in a known location in the genome- there are landing sites which are defined and we know that they are viable when inserted here and know they will be expressed- not in a highly chomatinised region.

32
Q

what do you need to keep in mind when you are designing RNAi probes? how can this same concept be useful?

A

genes have different transcripts due to different alternative splicing. So many of the libraries sign probes that will knock down all of the splice variants by identifying gene regions that are common to all transcripts. But if you want to find out the different functions of the different transcripts, you can design probes which will target specific transcripts.

33
Q

what is the role of a ftz intron at the end of the RNAI cDNA construct in dros RNAi?

A

its increases the efficiency of the RNAi

34
Q

what are three drosophila libraries that are available?

A

vienna drosophila resource center, national institute of genetics, japan, transgenic RNAi project, harvard.

35
Q

what are the the two libraries that the vienna drosophila resource center used and what is the difference between them?

A

GD library and the KK library they differ in the transposons that they use the GD has “p-element” which gives you a random insertion and the kk library used a phiC31 which means all are inserted into a known location within the fly genome in a safe, efficient “landing site”

36
Q

which library accounts for multiple transcripts?

A

VDRC

37
Q

how many genes does the NIG-Fly library cover?

A

11909

38
Q

what is the construct of the VDRC?

A

p-element, 10 UAS sites, hsp70, homologues arms of RNAi probe with linker in the middle, ftz intron, poly A tail and mini white gene, p-elemt.

39
Q

what is the construct of the NIG-Fly?

A

UAS-hsp70, clone two inserted repeats with a heterologous oncogene (exon-intron-exon) which improves RNAi efficiency.. Guessing there are known primer sites in the construct too?

40
Q

how many genes does the harvard medical school library cover?

A

10108

41
Q

what kind of transposon does the harvard library use?

A

phiC31 so it integrates into known region

42
Q

what is a cool bonus about using the harvard library?

A

their constructs allow for alteration of the number of UAS repeated to generate a series of different RNAi expression levels.

43
Q

what is a quirk about the harvard medical school RNAi library?

A

instead of using long dsRNA they use short dsRNA which work during oogenesis unlike normal long dsRNA (valium 10 based vectors) - short hairpins.

44
Q

how can you boost RNAi?

A

boost dicer, increase temperature and boost UAS

45
Q

what is a valium 20 based vector?

A

a short strand of dsRNA used by the harvard RNAi library which produce short hairpin RNAs not long ones.

46
Q

describe a screen that looked at wound healing using RNAI in flies?

A

they made wounds into fly larvea that normally takes 24 hours to repair in wild types. They screened for gene which are responsible for this wound healing. They used a red reporter driven by gal4/uas to stain nuclei and stained membranes with anti fasciclinIII to visualise the wound well. They were able to observe thephenotye of wound closure without dissecting ( saves a lot of time). They used 190 different RNAi strains from NIG-fly library. they found lots of genes in involved in this

47
Q

describe a genome-wide behavioural screen using the NIG fly RNAi library..

A

They used genes from the NIG fly RNAi library using 6000 strains and crossed with a strain expressing a neuronal sub-type specific GAL4 driver (in PDF neurons) and then they looked for weird circadian rhythms- found strip.

48
Q

what is miRNA?

A

the naturally occurring double stranded RNA that is present, but people aren to 100% sure what they do

49
Q

how did people try to wok out what the role of miRNAs were?

A

they wanted to find what the targets of the miRNA are and what happens when they dont find these targets. they create miRNA sponges. this is a construct that contain many perfect binding sites for the miRNA and when expressed (by the UAS system) they will sponge up the miRNA and will titrate these miRNAs away from the nucleus where they normally work.an miRNA sponge was made that soaked up 141 miRNAs, . the construct has a reporter (cherry) so you would know where it was being expressed. they increased the number of sponge copies (2) to increase efficacy. They then checked for expression of miRNAs so see if they are down regulated when comapred to a scrambled sponge construct (control). some were and some weren’t. they showed that the expression of the sponge was able to rescue the expression of the targets (see paper). The found that there were eye development defects etc.

50
Q

how many miRNAS are there in flies?

A

200

51
Q

how can you use the Gal4/UAS system to over express genes? why would you want to do this

A

instead of having reporter or RNAi, you can have an extra gene copy to see if you can rescue a mutant to prove the role of this gene in the process of interest. you can also look at the effect of dosage. you can express extra copies in the native tissue or in different tissues- induce eyes in fly legs etc.

52
Q

how can you use UAS for over expression?

A

can use a library and over express many genes to see the effect and look at function. and you can drive over expression in different areas of the embryo and in different points in development

53
Q

describe the entire process of making a transgenic fly.

A
  1. You have two parent flies that are mutant for the white eye gene 2. You cross these and make a fertilised egg. 3. You insert your gene of interest with its promoter (Gal4 with tissue specific promoter or UAS site with hsp downstream), and a reporter gene with its promoter, into a transposable p-element. This p-element is then injected into the fly egg into the area of the germ cells and helper plasmic containing transposes (under standard promoter?) that isn’t integrated is also injected. 4. the transposase then interrogates the P-element into the germ cells of the F1. F1. You can then cross the F1 which will have het of the p-element in the germ line and the F2 will have a red eyed fly that expresses the gene of interest too (Gal4 or UAS)
54
Q

how would you actually increase UAS or DICER expression with your RNAi?

A

increase the expression of UAS in the plasmid which is going into one fly and gal4 in the crossing fly