deck_4974882 Flashcards
(108 cards)
1
Q
What the main model organisms?
A
Yeast, plant (arabidopsis), c.elegans, drosphila melanogaster, zenopus, zebrafish and mice (musculus musculus)
2
Q
List of the order of genetic similarity to humans of the main model organisms.
A
Mice, xenopus, fish, fly, c.elegan, yeast, e.coli
3
Q
what percentage of coding DNA is different in mice compared to humans?
A
10%
4
Q
what percentage of coding DNA is different in fish compared to humans?
A
25%
5
Q
what should be kept in mind when studying the role of regulatory non-coding DNA in your model organism in relation to human application?
A
differences in non-coding DNA greatly, a lot more than protein DNA, for example there is an average change of 50% of non-coding DNA when compared to primates. It is better to use mice for such experiments as closely related.
6
Q
what makes a good model organism?
A
- short-breeding cycle - large brood size- high relating freqeuncy - robustness- simple feeding and other maintenance requirements- ability to store special strains in an inactive state- history (linkage maps, mutant collections, genome sequence) - molecular techqniues (transgenesis, targetted mutations) - you can do gene targeting and targeted mutagenesis
7
Q
what type of study is normally done in zebrafish?
A
development, disease, behaviour
8
Q
what type of study is normally done in mice?
A
behaviour, optogenetics, brain imaging, genetics, obesity
9
Q
what type of study is normally done in mice?
A
- cell signalling, nervous system study
10
Q
give two examples of model organisms not mirroring the behaviour of genuinely wild type animals?
A
- wild mice are more aggressive and less maternal instincts- c.elegans in the wild form clumps in response to high oxygen concentrations
11
Q
Why are c.elegans good for Knock out experiments?
A
there are libraries containing a strain expressing a KO for every c.elegan gene (save this)
12
Q
why list 9 reasons why c.elegans are a good model organism
A
- easy to maintain - short life cycle (around 3 days)- small number of cells- around 1000 somatic cells - transparent - fully cell-lineaged- detailed anatomy and full connective - genome fully sequenced (first animal)- ease of transgenesis - forward and reverse RNAi genetics
13
Q
why are c.elegans good to study development in?
A
- there re conserved genes in worms and in other organisms (transcription factors, homeodomains, bHLH, miRNAs, signalling pathways such as Notch, Wnt and TGF-beta)-There are conserved mechanisms: cell autonomous dell fate specification (cascades of TFs and miRNAs), cell non-autonomous signalling (tissue patterning, lateral inhibition)- cell lineage is invariable in the worm so you can ablate cells and look at inductive vs autonomous development .- because worms are transparent so can track lineage the entire time translational and transcriptional reporters can be used.
14
Q
how can c.elegns be stored for a long time?
A
in the dater state- dont need to be fed
15
Q
how many neurons do c.elegans have?
A
302
16
Q
how many glial cells to c..elegans have?
A
56
17
Q
how many morphological classes of neurons are there?
A
118
18
Q
what is considered the brain of the worm?
A
the nerve ring
19
Q
why is having a connectome not hugely important?
A
you want to know the precise interactions between the components, are the signals positive or negative long range or short range?
20
Q
why is the c.elegan being transparent a good thing?
A
- because worms are transparent so can track lineage the entire time translational and transcriptional reporters can be used.
21
Q
when are transcriptional reporters used? what are their down falls?
A
- to monitor gene expression or for cis-regulatory analysis (identify enhancer regions) - not all mRNA is translated- can’t be used to look at protein localisation
22
Q
when are translational reporters used?
A
- monitor gene expression - monitor protein localisation - cis regulatory analysis - over-expression or mutant rescue
23
Q
how do you make translational reporters in the c.elegans and how are they put into c.elegans in particular?
A
you produce a DNA construct containing the gene of interest and its regulatory regions upstream of it. Then you produce an overhanging sticky end which is complimentary to another construct’s end that contains the a gene for a reporter such as GFP with a 3’ unc- 54 UTR on the end of it. SO that they can anneal to each other and act as primers to each other during PCR. These can then be injected into the gonad for form an array (mini chromosomes) that is inherited in a mosaic fashion (can be generated in a week). They can be integrated into the genome for mendelian segregation using gamma/ uv-irradiation (how?)- cam also bombard using gold particles into the genome (how?)
24
Q
when can a mini chromosome be used in c.elegans and for what?
A
They can be used to study cell lineages- if you have a construct that has a fluorescence reporter downstream of a universal promoter and inject, it will be inherited mosaically so one cell will inherit fluorescence and the other won’t after division.
25
what is a fosmid construct and why is it used?
- In order to ensure that the gene of interest that is being inserted into the worm genome is being authentically regulated, a fosmid construct. This fosmid can contain a bicistronic region. A GFP can be inserted after a gene within a fomid but due to the intercistronic region, the primary transcript will undergo trans-splicing. This will result in the protein localising to the normal site while the GFP can undergo nuclear localisation (if has an NLS) and allows for cell identification.
26
what can be carried out in c.elegans?
forward genetic screens ,genetic dissection of developmental pathways (epistasis), mapping and cloning, reverse genetics (RNAi)
27
how many chromosomes does the fly have?
4
28
what percentage of human disease genes have genes in the fly?
60%
29
how many of the fly genes have homologues in mammals?
more than half
30
why are flies good for tracking recessive genes?
there is no recombination in male flies so you can associate a recessive gene to a visible marker (such as curly wings or white eyes) and you know there will not be recombination between this (but does it not matter about the females?- maybe if passed on from father to and only males used then it doesn't|)
31
what are two methods of tracking recessive alleles un flies?
- no recombination in males- using balancers
32
explain how balancers can be used to track recessive mutations
you can use a balancer which has many inversions on in which prvent recombination. Then have a recessive lethal allele on the balancer and a dominant visual trait so that you can locate the balancers. SO you can cross a mut/bal with a mut/bal, the offspring will be straight winged double mutants or curly winged het mutants. If there are none without curly wings then you know that the mutation is recessive lethal
33
how can you use balancers to determine if mutations are recessive lethal?
have a recessive lethal allele on the balancer and a dominant visual trait so that you can locate the balancers. SO you can cross a mut/bal with a mut/bal, the offspring will be straight winged double mutants or curly winged het mutants. If there are none without curly wings then you know that the mutation is recessive lethal
34
are balancers specific to flies?
no but they are most widley used in flies
35
describe how p elements can be used for fly transformation
1.Clone the P element into a plasmid and transform and grow this in bacteria.2.Eliminate the P transposase and replace it with your gene of interest.3.Microinject the posterior end of an early-stage (pre-cellularization) embryo with DNA coding for transposase and a plasmid with the reporter gene, gene of interest and transposase recognition sequences.4.Random transposition occurs, inserting the gene of interest and reporter gene.5.Once the gene of interest has been inserted it is no longer mobile because it cannot produce its own P transposase.6.Grow flies and cross to remove genetic variation between the cells of the organism. (Only some of the cells of the organism will have been transformed. Hopefully, some of these transformed cells end up in the germ line. A transformed gamete will give rise to an organism with no variation between its cells).7.Look for flies expressing the reporter gene. These carry the inserted gene of interest, so can be investigated to determine the phenotype due to the gene of interest.
36
describe how p elements can be used for mutagenesis
Insertional mutagenesis[edit]1.Microinject the embryo with DNA coding for transposase and a plasmid with the reporter gene and transposase recognition sequences (and often the E. coli reporter gene and origin of replication, etc.).2.Random transposition occurs, inserting the reporter gene randomly. The insertion tends to occur near actively transcribed genes, as this is where the chromatin structure is loosest, so the DNA is most accessible.3.Grow flies and cross to remove genetic variation between the cells of the organism (see above).4.Look for flies expressing the reporter gene. These have experienced a successful transposition, so can be investigated to determine the phenotype due to mutation of existing genes.
37
list the types of mutations that can arise from using p-elements
Possible mutations:1.Insertion in a translated region => hybrid protein/truncated protein. Usually causes loss of protein function, although more complex effects are seen.2.Insertion in an intron => altered splicing pattern/splicing failure. Usually results in protein truncation or the production of inactive mis-spliced products, although more complex effects are common.3.Insertion in 5' (the sequence that will become the mRNA 5' UTR) untranslated region => truncation of transcript. Usually results in failure of the mRNA to contain a 5' cap, leading to less efficient translation.4.Insertion in promoter => reduction/complete loss of expression. Always results in greatly reduced protein production levels. The most useful type of insertion for analysis due to the simplicity of the situation.5.Insertion between promoter and upstream enhancers => loss of enhancer function/hijack of enhancer function for reporter gene
38
once you have used a p-elemnt to induce a mutation, how can you map where in the genome the p-element has inserted?
Using a process called reverse PCR. This involves you inserting a primer site into your p-element which will allow you to PCR amplify. These known sequences of DNA will be either side of a restriction site that you have put into your p-element. So once, you p-element has inserted, you use restriction enzymes to cut up all of the genome and make linear fragments. Then you ligate up using the sticky ends to make circular DNA. Then you use your known restriction site inside you p-element and apply the restriction enzyme so that you circle of DNA of interest is linear with your known DNA seuqneces of the p-element at each end then yu can make primers of these ends and PCR amplify and then sequence the PCR product to find the gene or area of DNA in which the p-lement has been inserted.
39
why are zebrafish a good model organism?
- vertebrate so closely related to humans - rapid development-available in the lab all year round- optically transparentexcellent genetics- forward, reverser and transgenic- excellent embryology - easily injected- easy to make mosaicsconservation of gene function
40
what are the downfalls of flies?
??
41
what are the downfalls zebrafish??
- Don’t have good inbred lines- because seem to get bad fecundity when inbreed but just because hasn’t been used a model organism for a long time. This is bad because there is always going to be some genetic background which means that sisters and brothers are going to be different because the parents aren’t isogenic - have gene duplications because of genome duplication events
42
what are the advantages of using zebrafish for genetics?
- unlimited supply of embryos- accessibility - optic clarity- imaging - in vivo cell manipulation techniques from early to late development - genetic and genomic techniques- unlimited mutants- cost - more genes?
43
what is the gene knock-down technique in zebrafish?
Morpholinos
44
How do morphosinos work?
they bind to the parts of the mRNA and stop it from being translated. SO you can target an oligo to the AUG site of the mRNA
45
why would you want to use a moprholino in zebrafish?
If you wanted to do a knock down test to look for gene function in processes. Or if you could see if the knock down of a candidate gene function phenocopies the genetic mutation. You can carry out ubiquitous gene specific knock-down or mosaic knock-down by only injecting into specific areas.
46
what is a draw back of morpholinos
they have to be injected into the embryo so they are mosaic- not every cell affected.
47
what is a gain of function techqniue in the zebrafish?
you can inject RNA or DNA in a ubiquitous manner or in a mosaic manner.
48
once you have a candidate gene for a function, how can you consolidate the role of these gene (2)?
you can use a knock down technque to see if it can phenocopy the mutant or you can use gain of function technique to see if DNA or RNA expression of the candidate gene can rescue the mutant
49
what are the drawbacks of using the injection of DNA or RNA into the zebrafish to cause gain or loss of function effects?
they are not integrated into the DNA. This means that you can't make stable lines from these animals
50
how can you make stable transgenic lines with zebrafish?
You inject transposes and a plasmid containing DNA with a transposon construct (told sites either side of gene of interest- such as a reporter) then you inject this into the fertilised egg and the transposes will insert it into the genome of the cells of the embryo will all incorporate the plasmid hopefully. However, if you dont get this at the start of development then you will get a mosaic and can't guarentee a mendelian ratio of inheritence if not all of the germ cells have inherited the construct
51
if you wanted to make a reporter transgenic line and you wanted to ensure that the reporter represented the expression of the gene as interest as closely as possible, how would you do this?
you would use a BAC (bacterial artificial chromosome), these are large amount of DNA that should contain all of the regulatory regions of your gene. these can then be inserted into your genome ??
52
what are the use of dual BACS?
??
53
what are the drawbacks of BACs?
the larger piece of DNA the harder it is for them to be integrated into the genome?
54
how can you make chimeras in zebrafish? (2)
1. you can make a transgenic universal GFP fish. this can act as the donor. Cells can be taken from this fish and transplanted into an acceptor fish and then the fate of these cells can be viewed relative to the area in which they were derived in development. 2. you can also move cells from one side of the fish and place it into the other side of the embryo- such as when looking at the organiser cells etc- can induce two headed fish
55
how can you study the effect of recessive maternal effect genes, that cause the mother when mutant to die?
if you cant have a homo mum because the gene is lethal then you never have access to a homo mum (so the homos die even though the mum contributes early, because of later things) but you still want to see the developmental effects of the gene, you inject the donor embryo (which is homo mutant) with a marker that goes into the germ line. Then you can suck out these cells very early out of an inject into a wildtype fish that doesnt have any germ line cells (killed with morpholinos) so then you are putting these mutant germ cells into a surrgate that can actually survive, unlike the mutant recessive mother,
56
give an example of when the fish having 2 gene duplications has been a problem in research
autism (find jason's paper)
57
what is an advantage of using mice as a model organism for human studies
99% of human genes have a clear orthologue in the mouse
58
give an example of the difference between mice and human genetics
estimated >20% of human essential genes are non-critical in the mouse
59
what are the three approaches to genetically modifying mice and what are each of these approaches used for?
- microinjection of DNA into fertilised ouse eggs (transgenic mice): gene reg studies to characterise promoters, over expression or miss express studies on mutated or native proteins to induce or rescue a phenotype, expression markers (GFP, LacZ) - genetic manipulation of mouse embryonic stem cells and blastocyst/morular injections: knock-out and knock-in mice, gene trap strategies- genome editing
60
what is the process of DNA microinjection into the mouse egg?
the transgene is injected into the pronucleus of fertilised mouse eggs (BAC DNA or plasmid DNA containing regulatory elements and a gene of interest). The insertion into the genome is at random. it is injected into the male pronucleus because it is large. then the eggs transferred into the recipient mother and she produces a transgenic offspring- which is het?
61
what is the process of the knock in and knock outs?
using homologous recombination to and make a transcript with homologous arms and inject this into ES cells which have a maker (normally neo for resistance) and then inject into a blastocyst and transfer into a mother to form a chimeric mouse. then if incorporated into the gonad then fill produce a het knockout or knock in which you can use to make a homo mutant
62
what is the ROSA26-stop-reporter mouse?
you can use the area called the ROSA26 region which is redundant to insert genes. the ROAS26 promoter is also ubiquitously expressed so you can put your gene downstream but with a stop codon infront of it . you can use this region to insert reporter genes which can report the expression of a gene by using a cre loxp system. You can have a construct that has a SA and a loxp surrounding a neo, tpA (stop codon) upstream of a reporter gene, so that when cre is driven by gene of interest promoter, there is recombination and the reporter is transcribed
63
what are the advantages of using a ROSA26-stop-reporter mouse rather than a nuclear injected reporter construct?
you know where it will be inserted so dont risk being inserted into unwanted harmful area or unexpressed hetereochromatin area
64
how can the tamoxifen, oestrogen receptor in mice?
you produce a mouse that expresses a cre attached to a oestrogen receptor protein with one that expresses a floxed ROSA26 reporter construct. then when you add tamoxifenit will enter the nucleus.
65
what can you use the ROSA26 area for?
- for reporters for normal cre- for reporters or gene exogenous gene expression driven by cre of tamoxifen - for cell death driven by floxed DTA
66
why is being able to do homologues recombination in mice really important for gene expression analysis?
if you can do it right you can get fully physiological control of the reporter- all of the promoter regions are already there
67
give an example which demonstrates the importance of being able to see where your gene of interest is being expressed by using full regulatory influence?
Sloan and Barres 2014 wanted to stop gliotransmission by driving a gene that did this in glial cells by using a glia "specific promoter" but they didn't realise that it is expressed in neurons too
68
what is in utero electroporation used for?
you can inject DNA in certain sites by using electroporation - certain areas of the brain- dont have to do genetics in the one cell stage- also works in the fish.
69
what are retroviruses?
the genetic material of retroviruses is in the form of RNA molecules. When a retroviruses infects a cell it will introduces its RNA together with some enzymes, namely reverse transcriptase and integrease into the cell. The reverse transcriptase can produce a cDNA copy of itself and then the integrate will insert the DNA molecules into the cell
70
what are the problems with retroviuses?
they can insert randomly into the genome which can disrupt gene function
71
how can the insertion site of retroviruses be controlled?
they can be used in combination with a double stand break inducer.
72
what is an adenovirus?
viruses that carry their genetic matieral as double stranded DNA- the genetic material is not incorporated and is instead free in the cell.
73
what is an adeno-associated virus?
it is a small virus that can infect cells and will induce a negligible immune response. it can infect non-dividing cells and will integrate into the host genome- it normally integrates into the human chromosome 19- cool!
74
how could you make a translational reporter than didn't interfere with the proteins functioning and instead just labelled which cell was affected?
you add a IRES spacer and then a NLS attached to the GFP protein
75
go through the entire process of making a translational reporter in c.elegans
- you has a fosmid which you have ordered which you know contains the gene on interest - you know the sequence of the gene so you can make homologous arms on your GFP construct that will recombine din - your GFP construct contains 4 GFP genes, with a selection maker in the middle Galk surrounded by FRT sites. - you insert the fosmid and your recombining structure into a a yeast strain and grow on the medium galactose. - you then select those colonies that survive- you introduce FLP arabinose - then you will have strains elect dfor by growing on deoxy-galatcose cntaing your fosmid which you can purify out and inject!
76
how can transcription reporters be implemented in c.elegans?
via a plasmid which doesn't integrate and is inherited in a mosaic fashion or by actually inserting into the genome for a trasngenic line
77
how would you carry out a transcriptional reporter study using a transcription reporter that is in a plasmid?
Plasmids containing marker and target DNA are injected into the syncytial gonad arm. Injected DNA then forms an extrachromosomal array or minichromosome. Progeny that inherit the array display the marker phenotype in a mosaic fashion (?)
78
why are balancers used?
Balancers are most often used in Drosophila melanogaster (fruit fly) genetics to allow populations of flies carrying heterozygous mutations to be maintained without constantly screening for the mutations but can also be used in mice.[1] Balancer chromosomes have three important properties: they suppress recombination with their homologs, carry dominant markers, and negatively affect reproductive fitness when carried homozygously.
79
how would you use Homolgous recombination to find out where your gene of interest is expressed- you would want to do this to make sure your Cre is going to affect the right cells.
you can HR you GFP into a part of the protein you know won't interrupt the functioning into the ES cell and then inject into th blastocyst then the mouse mother
80
what should an organiser be able to do?
- form the dorsal anterior mesoderm - induce both the AP and DV axis- induce a secondary axis when transplanted - change in its inducing abilities over time
81
what is the difference between a BL transplanted from an early gastrula as opposed to from a later gastrula?
- it will form an entire axis early on but later it will only be able to for more posterior structures
82
what do the cells from the blastopore lip go on to form in the xenopus?
anterior endoderm, prechordal late, head mesoderm, notochord
83
where is head inducing tissue found in the early blastula?
in the dorsal vegetal region- the presumptive anterior endoderm- acts as the AVE
84
what do the AVE cells express in the mouse?
crescent, cerberus, frzb, otx2, hex- they are involved in head patterning
85
where is cerberus expressed in xenopus?
anterior endoderm
86
how does cerberus block BMP WNT and Nodal?
binds to them outside the cell
87
how is neural induction different in the chick than in the xenopus?
BMP inhibition can not replicate the actions of the node to induce neural tissue- they can stabilise sox 3 expression after 5 hours but that is it- there is a different mechanism for neural induction- involving FGF from the node and from the underlying hypoblast
88
how has the organiser beens how to not be required for neural tissue in xenopus and what has been proposed as a result?
a neural plate will still develop in frogs, birds and mice the organiser or node is excised during gastrulation, although chordin and noggin encode for neural inducing signals in zebrafish, double knock out does not prevent a neural plate from developing - therefore it has been suggested that neural fate is given before gastrulation- maybe involving the WNT signalling in the early dorsal patterning of the xenopus embryo
89
what pieces of evidence suggests that gene expression in the mesoderm may be influencing gene expression in the in the ectoderm?
several box genes int he notochord and in the pre-somatic mesoderm and ectoderm are at the same position along the axis.
90
where can hox gene expression not be found in the mouse and what is thought to act in their place?
it can't be found in the anterior most neural tissues such otx and em are expressed anterior to the hindbrain and these gene encode homeodomain TFs and specify th pattern of the neural plate
91
what is the simple two step process by which neural patterning is thought to occur along the neural tube?
mesoderm induces the antire overlying ectoderm to become anterior and then posteriorising signals transform the posterior parts of tissues after.
92
what are the posteriorising tissues thought to be?
FGf ants WNT3a
93
what are the two signals implicated in AP neural tube patterning ?
the first are the anterior signals thought to be noggin ad chordin, the second are the posteriorising such as FGF and WN3a
94
how do the node and the hypoblast act together to induce the AP axis along the neural tube in the chick?
the node emits FGF and posteriorisig signals, the anterior neural tissue must be protected from these signals- the anterior mesoendoderm is though tot achieve this as it moves anterior to the node after ingression and releases factors such as cerberus, frzb, dkk etc
95
what are the two organiser within the neural plate, what are their roles?
One of them is the anterior neural ridge, which lies at the junction between the prosencephalon and the anterior ectoderm, and is necessary for the maintenance of forebrain identity. The second one is the isthmic organizer (IsO), which lies at the junction between the midbrain and hindbrain, and is necessary and sufficient, for the development of mesencephalic and metencephalic structures.
96
what does the anterior neural ridge express?
FGF8, otx2, chordin, shh,
97
what is the Anterior neural ridge essential for? how has this been shown?
patterning of the telencephalon, removal of it in fish or mice will result in lack of forebrain expression of the telencephalon marker and gratifying an ectopic one into the diencephalon will induce telen marker expression
98
what is the role of the isthmus organiser? how was this shown?
found at the midbrain hindbrain junction, if you transplant it, it will induce the formation of an ectopic midbrain in the hindbrain
99
what is found in both the anterior neural ridge and the isthmic organiser?
FGF8
100
what is different about the neural induction in the chick and in the xenopus?
it doesn't occur by default in the chick- BMP inhibition is not sufficient- instead it is a multiple step process
101
what is the FGf signalling pathway?
FGF TK receptor -grb, SOS, RAS, RAF, MAPKK, MAPK, acts with TF
102
what does the prechordal plate underly?
the anterior forebrain
103
what does the notochord underly?
the ventral head mesoderm
104
what is the neural default model?
this proposes that the defualt model of neural induction in xenopus. this proposed that the default stae of the dorsal ectoderm was to develop as a neural tissue but this pathway is blocked by the presence of BMPS which promote epidermal fate. The role of the organiser so to lift this block by producing proteins that inhibit BMP actvity, the region of ectoderm that comes under the influence of the antagonist form the organiser act on ectoderm and cause it to form neural tissue. according to this model the signals from the organiser act on the ectoerm that lies adjacent to the organiser at the bedginning of gastrulaion. as gastrulation process, internalises cells continue to act on the ectoderm that now overlies them.
105
what does the elimination of one BMP antagonist do compared to all 3?
when you remove 1 no effect but when you MO against them all- ventralise
106
explain how FGF has been implicated in neural induction the xenopus?
- neural development in xenopus also required FGF when even BMP is lifted by noggin or chordin. FGF is produce by cells in the blastula that are thought to act as early neural inducers. - it has also been shown that apparently spontaneous differentiaiton of explanted newt ectoderm into neural tissue is caused by activation to the MAPK pathway, this occurs in the FGF signalling pathway and treatment of explanted newt ectoderm with small molecule inhibitors of the pathway prvents neural differentiation. the ras-mapk pathway is also activated when xenopus extodermal cells are dissociated which might explain as noted ealrier hwy this dissociation forms neural fate so easily. induction of the neural plate therefore needs not only the antagonism of BMP signalling but also FGF siganlling (like in chick!)
107
how is FGF signalling though tot act in the neural patterning of the chick?
the effects of FGF neural induction may result from a direct inducting effect, activated MAPK can interefere with BMP signalling by causing an inhibitory phoshprylation of smad 1 which is part of the intracellualr BMP sgnalling pathway.
108
describe the 'qualitative' and 'quantitative' components of neural plate AP patterning
qualitative= the mesoderm secrets different antagonists at different points along the AP axis- it secretes BMP, Nodal and WNT inhibitors at the very anterior. But then only BMP antagonists are secreted more posteriorly, thought to induce spinal cord. quantitative: WNt/b-cat signalling along the AP axis is the neurula acts int a posterior to anterior graded manner, injected animal cap explants with different levels of NWT produces different posterior markers. FGF is also a posteriorising factor in a gradient.
