Laura Moody Flashcards
Transcriptional reporters
- promotor + tag + terminator
- transform the construct into plant
- where the promotor is active, you drive expression
- tells you which cell types have the active promotor
Translational reporters
- promotor + sequence + tag + terminator
- drives expression of gene fusion (you need the start codon intact; you have to remove the stop codon)
- gives protein localisation
What assumptions do you make when using transcriptional and translational reporters?
- does not interfere with cellular processes
- not affected by external factors
- (does not affect folding of the protein)
- (does not affect PTMs)
How do you predetermine the sample size using stats?
power analysis
How might you go about testing whether it is possible for a cell to express a protein? [as proof of the functionality of transcriptional reporter]
- attach it to an NLS
- can be visualised easier - you know where to look
- if it’s not highly active in the cytosol you might not be able to see it
- easy to quantify
- compare between cells
Why might you use a combination of transcriptional and translational reporter?
- it will tell you about the autonomy of the cellular behaviour
- is the protein excluded?
- are there any PTMs?
GUS
visual resolution of enrichment; are the Figures clear enough
When analysing figures
- look at what they actually show
- have they over-interpreted?
- are there any questionable labels?
- is there anything that just isn’t addressed?
Model organisms?
- can you use a single cell?
- can you transform?
- is there genomic data?
phylogeny
the only way to determine how many genes, and their relatedness
Reporters
- will not always have the same resolution
- we can assume that the chosen reporter has the best resolution for this context, although the use of other reporters might be useful
- e.g. are the images blurred?
- if they are super-clear, say it is a high resolution reporter
Why would you use GFP/citrine over GUS?
- they are fluorescent reporters, not colorimetric
- fluorescence microscopy (confocal laser scanning/two-photon) over light microscopy
- you can’t do live cell with GUS, because it requires fixation and staining; easier to do in cells that you can’t transform with GFP
How can you quantify under fluorescence microscopy?
- image based quantification using region-of-interest analysis (software-based; manually define the nucleus using DAPI; nuclear-to-cytoplasmic ratio)
- co-localisation with DAPI using Pearson’s correlation coefficient
- flow cytometry with DAPI
- Western blotting after cell fractionation, normalised to nuclear and cytoplasmic controls
- live-cell imaging (aka time-lapse microscopy); software-based
GUS resolution
- can’t do live cell imaging, so it’s not as good at tracking individual cells
- further research?
Using different reporters
- will have different stabilities in different model systems
- you can assume that the reporter chosen by the researchers is suitable, but perhaps helpful to test others?
who decided that 0.05 was an appropriate degree of significance?
Fisher
further research
- live cell imaging using GFP!
How do you test if a protein is mobile?
fluorescence recovery after photobleaching
Why is arabidopsis a good model organism?
- Small Genome & Well-Annotated Genetics
- Short Life Cycle & High Seed Production
- Ease of Genetic Manipulation
- Small Size & Rapid Growth
- Large Collection of Mutant & Genomic Resources
- Relevance to Other Plants
Why are mice a good model organism?
- Genetic Similarity to Humans
- Short Generation Time & High Reproduction Rate
- Fully Sequenced & Well-Annotated Genome
- Powerful Genetic Tools (transgenic, KO/KI, humanised)
- Well-Established Disease Models
- Cost-Effective & Easy to Maintain
- Ethical & Regulatory Infrastructure
Why are zebrafish a good model organism?
- Rapid Development & High Reproductive Rate
-Transparent Embryos - Genetic Similarity to Humans
- Well-Developed Genetic Tools (CRISPR, transgenics, mutants)
- Whole-Organism Drug Screening
- Regenerative Capabilities
-Cost-Effective & Space-Efficient
Why is C. elegans a good model organism?
- Simple Anatomy & Small Genome
- Transparent Body for Live Imaging
- Short Life Cycle & High Reproductive Rate
- Powerful Genetic & Molecular Tools (RNAi, Cas9, CRISPR, transgenics)
- Nervous System & Behavior Studies
- Aging & Longevity Research
- Space-Efficient & Cost-Effective
Why is Ciona intestinalis a good model organism?
- Closest Invertebrate Relative to Vertebrates (urochordate)
- Highly Conserved Developmental Pathways
- Small & Simple Genome
- Transparent Embryos for Live Imaging
- Rapid & External Development
- Genetic & Molecular Manipulation (CRISPR, electroporation)
- Cost-Effective & Easy to Maintain
Limitations of arabidopsis
It is not a crop
Why is Drosophila a good model organism?
- Small Genome & Genetic Similarity to Humans
- Short Life Cycle & High Reproductive Rate
- Powerful Genetic Tools & Mutant Collections
- Low Cost & Easy Maintenance
Why is Saccharomyces cerevisiae a good model organism?
- Simple Genetics
- Rapid Growth & Reproducibility
- Powerful Genetic Tools (CRISPR; KO via HR; plasmid transformation)
- Cost-Effective & Space-Efficient
