Lecture 2- Techniques in Developmental Neurobiology Flashcards
1
Q
What are the six model organisms used in neurobiolgical research?
A
- C. elegans
- Drosophila (fruit fly)
- Zebrafish
- Xenopus (African clawed frog)
- Chicken
- Mouse
2
Q
What are the advantages/disadvantages of using C. elegans as a model organism?
A
Advantages:
- easy to use, fast life cycle
- good for live imaging
- cheap
- only 302 neurons
Disadvantages:
- invertebrate, no cortex or neural crest
- evolutionary distance
- simple nervous system
3
Q
What are the advantages/disadvantages of using Drosophila as a model organism?
A
Advantages:
- fast life cycle
- powerful genetic systems
Disadvantages:
- invertebrate, no cortex or neural crest
- evolutionary distance
4
Q
What are the advantages/disadvantages of using Zebrafish as a model organism?
A
Advantages:
- fast life cycle
- powerful genetics
- good for imaging (embryo is see-through)
Disadvantages:
-evolutionary distance
5
Q
What are the advantages/disadvantages of using Xenopus (the frog) as a model organism?
A
Advantages:
- easy access to embryo
- large early embryo
Disadvantages:
-evolutionary distance
6
Q
What are the advantages/disadvantages of using chicken as a model organism?
A
Advantages:
- easy access to embryos
- cheap (eggs)
Disadvantages:
-difficult to generate genetic models
7
Q
What are the advantages/disadvantages of using mice as model organisms?
A
Advantages:
- mammalian, closer evolutionarily
- powerful genetic model
Disadvantages:
- slow to generate models
- expensive
8
Q
What are the main techniques used in neurodevelopmental research? (list 5)
A
- Fundamentals: immunohistochemistry, Western blotting and in situ hybridisation
- Physical ablations/transplants
- Transient/permanent/inducible genetic modifications
- Live cell visualisation and tracking
- Cell migration assays
9
Q
What is immunohistochemistry?
A
- the process of detecting antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues
- use of antibodies to label target proteins for visualisation via histological or fluorescent tags
10
Q
What information does immunohistochemistry provide? (4)
A
- positional information about protein location
- used to look at proteins inside the cell
- doesn’t work well on secreted peptides as they are dilluted
- not a quantitative method
11
Q
What are primary and secondary antibodies?
A
-groups of antibodies based on whether they target a target of interest directly or target another (primary) antibody that, in turn, is bound to a target of interest.
12
Q
What molecules are used in immunohistochemistry as binding and reporter/marker molecules?
A
- binding molecules are either primary or secondary antibodies
- reporter molecules are mostly chromogenic or fluorescent depending on mode of detection (often fluorophore is used)
13
Q
What is needed to achieve double/triple or four-colour immunohistochemistry?
A
-suitable antibodies, fluorophores, lasers (to excite fluorophores) and filters (for detection of different wavelengths) must be available
14
Q
What is the CLARITY imaging technique?
A
- fluorescent labelling and imaging of the whole brain
- technique where lipids are washed away with electrophoresis-mediated detergent while proteins are bound to “scaffolding” hydrogel monomers (often acrylamide)
- formaldehyde facilitates binding of the proteins to the scaffolding and heat is needed for establishment of the links
- imaging itself is accomplished by immunostaining using antibodies with fluorescent tags
- after each staining antibodies can be removed and new applied eventually creating a whole brain image (theoretically)
- it is a very slow and expensive technique thus not used commonly so far
15
Q
What is the Western blot technique?
A
- used to detect specific proteins in a sample
- uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide
- may need to denature the proteins, gives them negative charge
- proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein
- it is quantitative as can use the amount of housekeeping genes as a way of controlling for the amount of protein
16
Q
What is in situ hybridisation used for?
A
- uses a labeled complementary DNA or RNA strand to localise a specific DNA or RNA sequence in a portion or section of tissue (in situ)
- uses tagged short single stranded DNA or RNA (oligomer) to bind to mRNA
- reveals expression patterns of a gene
- sensitive technique
- can be semi-quantitative
- REMEMBER: staining does not neccessarily mean that there is a functional protein produced from the RNA (not clear why)
17
Q
How does in situ hybridisation differ from immunohistochemistry?
A
-immunohistochemistry localises proteins in tissue sections whereas in situ hybridisation localises specific DNA or RNA sequences
18
Q
What is the process of in situ hybridisation?
A
- the probe is labelled RNA (can be DNA too), it hybridises to the target sequence at elevated temperatures
- then the excess probe is washed away
- the probe labeled with either radio-, fluorescent- or antigen-labeled bases is localised and quantified in the tissue using either autoradiography, fluorescence microscopy or immunohistochemistry
- if you are using a phosphate bound antibody you need to add substrate for the enzyme
19
Q
What is neural crest ablation?
A
-where part of the neural crest in a developing embryo is removed and can be replaced by neural crest obtained from a different embryo (then it is neural crest transplant)
20
Q
What is the quail/chick chimeric model?
A
- most famous example of interspecies grafting
- part of the quail neural crest is transplanted into the chick embryo
- the result is visible due to observable differences between quail and chick cell nuclei
- nowadays difference is observed by using the QCPN antibody that is quail cell specific
- this model allows for identification of sub-populations of cells contributing to specific neural crest derived structures
21
Q
What are the ways of genetically modifying developing embryos?
A
1. Viral infection
- vector: virus
- can be integrating (permanent) or non integrating (transient)
- model is mouse or chick
2. Transfection-
via a) electroporation
- vector: plasmid, BAC (bacterial artificial chromosome), Morpholino, siRNA
- model: all in vivo and in vitro models
- not permanent
or b) direct injection
- vector: plasmid, Morpholino, siRNA
- model: early zebrafish and Xenopus (frog) embryos
- not permanent
22
Q
What is transfection?
A
-the process of deliberately introducing nucleic acids into cells
23
Q
What is electroporation?
A
- uses the fact that DNA is a charged (-ve) molecule
- application of current across tissue causes DNA to move towards the +ve electrode and opens hydrophobic micropores within cell membranes (these are transient)
24
Q
What is a morpholino?
A
- a molecule that is used to modify gene expression
- short oligomer sequences that bind to mRNA
- function via steric competition
- Morpholinos block small (~25 base) regions of the base-pairing surfaces of RNA, binding of a morpholino post-splicing prevents translation of that protein (=knockout)
- used as research tools for reverse genetics by knocking downgene function
25
What is siRNA?
- class of double-stranded RNA molecules, 20-25 base pairs in length
- siRNA acts in the RNAi pathway where it interferes with the expression of specific genes with complementary nucleotide sequences.
- siRNA functions by causing mRNA to be broken down after transcription, resulting in no translation
- highly evolutionary conserved mechanism of genetic regulation
- blocks transcription of target genes
26
Where was siRNA discovered?
-in C. elegans where the short hairpin RNA sequences were used to regulate gene expression
27
What is RISC (RNA-induced silencing complex)?
a multiprotein complex that incorporates one strand of siRNA and uses it as a template for recognising complemetary mRNA
-upon recognition it one of the RISC enzymes is activated (argonaute) and cleaves the RNA
28
What types of transgenic modelling can be done? (4)
1. Knockout
2. Knockin
3. Conditional
4. Inducible
29
What is knockout (transgenic models)?
-loss of gene function
30
What is knockin (transgenic models)?
- method that involves the insertion of a protein coding cDNA sequence at a particular locus in an organism's chromosome
- the difference between knock-in technology and transgenic technology is that a knock-in involves a gene inserted into a specific locus, and is a "targeted" insertion
- e.g. Fluorescent reporter for cell/ lineage tracing
31
What is a conditional gene knockout?
- a specific target gene is eliminated from a single organ in the body of an experimental animal, rather than the whole body, as conventional gene knockout technology would entail. It can be applied to eukaryotic and prokaryotic systems
- tissue and/ or time specific expression
32
What is an inducible gene knockout?
-expression is controlled by external factors
33
How do we get mutant mice?
- some spontaneous models exist with naturally occurring deletions or null mutation
- the desired knockout is usually engineered using DNA recombination in embryonic stem cells
- can also do non-targeted insertions
34
How do you perform a transgenic experiment? (5)
1. Isolate embryonic stem cells from inner cell mass of blastocyst
2. Transfect with targeting construct
3. Recombination events
4. Select with resistance marker
5. Inject cells into new blastocyst
35
What types of genes are good to choose to promote expression of the transgene? (4)
1. Housekeeping gene (e.g. ß-Actin: it is in all cells and use GFP as marker)
2. Tissue specific promoter (e.g. ß-Tubulin: in all neurons and use GFP as marker)
3. Inducible promoter, addition of molecular switch (e.g. TET on/off system)
4. Other inducible system- using tamoxifen to activate conditional gene deletion with Cre-lox system (Cre-ER fusion)
36
What happens in conditional transgenic mice?
- tissue-specific and inducible modification of gene activity, can delete gene or part of gene to inactivate or activate a transgene by deleting a "STOP" signal
- most commonly Cre-loxP system
- can use a fusion protein of Cre fused with a modified estrogen receptor that binds tamoxifen and, when tamoxifen is bound, transports Cre into the nucleus to perform the gene deletion of a "floxed" allele (=flanked by loxP sites)
37
What is Cre recombinase?
- site-specific enzyme, recognizes pairs of loxP sequences
- genetically recombines the DNA sequence
38
What does in-diet tamoxifen treatment induce?
-Sez-6 gene deletion in forebrain principal (excitatory) neurons that express calcium-calmodulin kinase IIsalpha
39
What happens in Sez6 conditional knockout?
-Sez-6 protein is not detectable in hippocampal pyramidal neurons
40
What is the SLICK technique?
- single-neuron labeling with inducible Cre-mediated knockout (SLICK), is achieved by coexpressing a drug-inducible form of Cre recombinase and a fluorescent protein in a small subsets of neurons, thus combining the powerful Cre recombinase system for conditional genetic manipulation with fluorescent labeling of single neurons for imaging
- tamoxifen-inducible Cre deletion in small subset of neurons, identified by Yello Fluorescent protein (YFP) expression
41
What does live cell imaging allow for?
- real-time analysis of migratory behaviour
- speed, directionality, transient cell-cell interactions can be measured
- can also use "photoconverted" cells to track discrete cells/populations
42
What types of cell migration assays are there? (2)
1. In vitro assays with cultured cells, either primary (e.g. neurons isolated directly from embryonic mouse brains) or immortal transformed cell lines (e.g. from neuroblastoma) can be cultured
2. ex vivo preparations (e.g. brain slice cultures)
43
What are the advantages of cell migration assays? (4)
1. parameters can be easily changed e.g. test factors can be added to culture medium
2. precise environment can be defined
3. easy imaging
4. 2D system e.g. growing neurons and other cell types adhere strongly to glass cover slips or plastic culture dishes and grow fairly flat
44
What are the disadvantages of cell migration assays? (3)
1. Primary cultures: different cell types mixed, also topography of connections (e.g. between different types of neurons or different brain regions) is disrupted as neurons grown in dissociated culture (3D arrangements not preserved)
2. Artefacts can arise from keeping cells in culture; also slice cultures change over time and do not resemble the in vivo situation
3. Can be difficult to maintain organ shape- challenging for imaging and analysis
45
What are cell migration/neurite outgrowth/ growth cone turning assays used for?
- allow for testing of putative attractive or repulsive factors separately
- may use primary cultured neurons or tissue/organ explants e.g. Dorsal Root Ganglion DRG axon outgrowth
46
What are the things you should consider when designing experiments to test a particular hypothesis?
- choose the best organism/model for the job, consider how relevant will your findings be to human biology/pathology
- use a spectrum of different techniques to address the question in different ways (biochemistry, cell biology, genetics, physiology, behaviour, bioinformatics)
47
What does looking at biochemistry tell us in an experiment?
-how do protein levels change during development
48
What does looking at cell biology tell us in an experiment?
-in which cell types, which sub-cellular compartments is the protein located
49
What does looking at genetics tell us in an experiment?
- Are mutations in the gene associated with disease? What are the consequences of inactivating the gene?
- points us to the function of the protein
50
What does looking at physiology tell us in an experiment?
-how is neuron/brain activity altered by loss of function, gain of function, mutant versions?
51
What does looking at behaviour tell us in an experiment?
-e.g. learning and memory tests, motor tests, social tests
52
What can looking at bioinformatics tell us in an experiment?
-global analysis of gene expression changes, gene networks
