Experimental techniques Flashcards
Explain how Genome-Wide association studies work
GWAS- Can help find correlations between genetic differences and phenotypes. You sequence many people, some with a disease and some without, then you can look at what the genetic variation between wt and diseased subjects - help to isolate a potential gene that is causing a problem. However, they do not show you where and when the gene is expressed and doesn’t show you how the protein coded contributes to the disease. Data from these studies are shown on Manhatten plots. 100,000 genomes project by sanger institute - helped identify genetic variant which correlates to T2D susceptibility. - but glucose homeostasis is complex so to find out where the gene is acting you could do in-situ hybridisation to look at its expression (found in pancreas) - Then do loss/gain of functions to look at effect.
Explain how the Cre-LoxP recombinase system works and what it is used for.
Used to make tissue-specific knockouts. You can also control when you KO the gene so is good for looking at mutations that are usually embryonic lethal. In one line of the animal, you flank the gene you want to knockout with LoxP sites (must be the animal’s endogenous gene). In another line, you identify a gene that governs the tissue/ time-specific expression of a gene - whatever is downstream of that promoter will only be expressed in that tissue at that time. You clone the coding sequence for Cre recombinase downstream of this promoter. Then you breed the 2 lines together, and where the tissue-specific promoter is expressed, Cre will cut out the gene flanked by LoxP sites to knockout that gene in that specific tissue. You can also sue a specific type of Cre that has an ERT2 site, which then is only active when you add tamoxifen. You can easily maintain the lines and mix and match them to cut out other genes in the same tissue of the same gene in different tissues.
Explain how FACS works and what it’s used for.
Fluorescence-activated cell sorting - uses a laser to sort cells labelled with different fluorescence tags (e.g. with reporters or antibodies) into different populations
How do you make reporter lines and why are they useful?
Reporter lines depend on the fact that genes are differentially transcribed as a function of the interactions of their promoter/enhancer and the cell-specific factors/activators. This means they are tissue and cell-type specific. You fuse the specific promoter to the coding sequence of genes that code for proteins that fluoresce when you shine UV light on them - e.g. GFP(eGFP) and Kaede which are green, or RFP/tomato which are red. You then make a transgenic line that stably incorporates the protein gene into the genome. You can follow the transgenic reporter over time in health, disease, after insult and after drug treatment. You can also use them to work out cell lineage. Large numbers of animals and reporter lines mean that animal models are used to powerful effect in the development of new therapies. The protein doesn’t fade overtime unlike immunohistochemial staining
What are some ways of analyzing cell proliferation in animals?
You can use one of 2 chemicals (EDU or BRDU), both are analogs of thymidine that have been modified to be visualised (either by antibody (BRDU) or chemical reaction (EDU)) - thymidine is an essential component of DNA (T nucelotide). If a cell is actively dividing it needs to replicate its DNA before it divides. SO when you add the chemicals to your model, it will incorporate it into its DNA when there is cell division. When cells are not dividing it will not be incorporated. You can then visualise and quantify how much proliferation there is
How can you use Cre-LoxP system for lineage tracing? What is lineage tracing used for?
Used for when you need to mark individual cells and their daughters permanently to trace what cells develop from/become. E.g. we know that GLAST promoter is in tanycytes (hypothalamic stem cells), so you add a cre ERT after the GLAST promoter in one of the lines. In the other line, you use a classic mouse promoter e.g. ROSA6 which contains a stop cassette but no active genes. You attach a GFP after the stop cassette and flank the stop cassette with LoxP sites. When you cross the lines (and add tamoxifen) the stop cassette is cut out and GFP is expressed in those specific tissues/cells. This causes a permenant genetic change that can also be found in the daughter cells (unlike if you just express GFP after GLAST because when GLAST is turned off e.g. when the cells start to differentiate you won’t be able to track them)
Explain how you culture embryonic stem cells initially and how you can check their pluripotency
You plate the inner cell mass on a layer of feeder cells (irridated stromal cells derived from later embryos which support ES cell growth) Once embryonic cells have divided a few times, you disaggregate them and replate them. Critical signals maintain cells in self-renewing, undifferentiated state and can replace feeders (includes LIF, BMP for mince and FGF2, TGFb for humans. Embryonic stem cells express the main pluripotency factors oct4, sox2 and nanog, and show no genes indicative of differentiation. A single cell can generate identical daughter cells. Genetically modified ES cells can be reintroduced into the blastocyst and contribute to normal development. e.g. ones that have GFP in.
What are the 2 types if in vitro differentiation and how do they work.
Target cell types are defined by the expression of the right markers and functionality.
- 3D - remove signals that keep cells in an undifferentiated state. Grown in aggregates (leads to embryoid bodies in the presence or absence of signals - EBs contain many undifferentiated cell types. Advantages - recapitulates more accurately they embryonic environment. Disadvantages- Difficult to observe/dissect the roles of individual signals.
- 2D/adherent - plays a defined number of cells on the right substrate/extracellular matrix. Remove signals that keep cells in an undifferentiated state. Grow in a defined medium with an appropriate amount of signals. Advantage: more tractable system (e.g. for live imaging), easier to test the role of specific signals. Disadvantages: loss of cell interactions that may occur in vivo
How does genetic ablation work?
Turn on a gene for nitroreductase, which is harmless to the cell on its own but makes them sensitive to a chemical (metronidiazole, so they die (you’re not ablating a gene, but using genes to ablate cells)
Explain how tunnel assays work and what they are used for.
Terminal deoxyribose transferase dUTP nick end labelling - Used to show that cells have gone through cell death. When cells go through apoptosis, they chop up their chromosomes into many small fragments. The DNA fragments is tagged at the end with Br (or other nucelotides) - and then an antibody specific to that nucleotide is used. Normal chromosomes only have a few ends so have low levels of fluorescence.
Describe another way other than Tunel assays that can be used to identify cell death.
Caspases are upregulated during apoptosis. Antibodies for caspases can be used as an alternative way to detect apoptosis.
What controls should you do for a western blot?
Positive and negative lysates (lysates from cells that are known for having/not having the protein you are detecting) - negative checks for non-specific binding and false positives. Loading controls - using antibodies for housekeeping genes to show that the level of lysate loaded is the same. Endogenous control lysate - loading a sample of the endogenous (non altered protein) when detecting a recombinant protein. Secondary antibody only controls - again to show non-specific binding.
What is the difference between a positive and negative control?
A positive control is when you expect a change. A negative control is when you expect no change.
How does a WST-1 assay work and what is it used to show?
The WST-1 assay protocol is based on the cleavage of the tetrazolium salt to formazan by cellular mitochondrial dehydrogenase. The amount of the dye generated by activity of dehydrogenase is directly proportional to the number of living cells.
How does CRISPR/Cas9 work and why is it used?
A guide RNA binds to a specific site on the DNA and tells the Cas9 enzyme where to cut. This enables you to cut out/add in a specific piece of DNA. DNA repair mechanisms then fix it. A precise and quick method of genetic engineering.
