Week 2 Flashcards
Why are animal embryos good for observing neural development
- Developmental mechanisms are evolutionarily conserved
- Many regulatory genes are similar
- Embryonic development is largely under genetic control
There are 2 fundamental types of reverse genetics, what are they and what are they used for? (Hint = think necessity / sufficiency)
- Gain of Function: To show a gene is SUFFICIENT for a biological process
- Loss of Function: To show that a gene is NECESSARY for a biological process
What are 2 common methods of Gain of Function transgenics? (think essay)
- Reporter transgenes: enables visualisation of specific cell types in a transgenic animal
- Tissue-specific expression of cre recombinase (for tissue-specific targeting)
What is a reporter gene? Give some examples
- Method to ‘mark’ a population of cells
- Example: Green fluorescent protein (can be observed in living tissue)
- Another example: LacZ - precipitates beta-galactosidase that can be dyed blue (but only in dead embryos)
What is CRISPR/Cas9 and what can it be used to make?
- RNA-guided system for making double-stranded breaks at precise position in DNA
- Makes transgenesis highly efficient
- Works in any species or cell-type
- Can be used in any cell type, including 1-cell zygotes
- Can be used to make either l.o.f or g.o.f alleles
How does CRISPR/Cas9 work?
- Cas9 enzyme is taken to correct sequence by a guide RNA with a complementary sequence
- Cas9 nuclease cuts both strands of DNA - creating a double strand break
Double stranded breaks an be repaired in 2 ways, what are they and how are they done?
- Non-homologous end joining (NHEJ): Repair mechanism based on speed rather than quality / accuracy. It directly re-joins broken DNA at the ends that were broken which may lead to lost bases if the break is staggered.
- Homology-directed repair (HDR): A more specific method that reduces chance of mutations. A DNA template or ‘homologous sequence’ accompanies the DNA-cutting enzyme so that the repair results in an altered or inserted gene
What are some drawbacks of gene knockouts? (2)
- Redundancy due to the presence of gene families
- Important gene null mutations are perinatal-lethal precluding analysis of gene function in adult life
How are genes in specific tissues targeted (easy)
Floxing mate
The developing cerebellum contains two progenitor zones. What are they and what type of neurons do they produce?
- Ventricular zone: Excitatory neurons
- Rhombic lip: Inhibitory neurons
What is Math1?
- A transcription factor expressed in the rhombic lip throughout embryonic development.
- Also known as Atoh1
A Math1-creER transgene was used in an experiment intended to observe the development of cells born in the rhombic lip. How did this experiment work in brief?
- Math1-creER transgene: contains a math1 promoter and a tamoxifen inducible cre-recombinase. This gene is localised to the RL
- Genetic fate mapping: Cre-reporter transgene used to give an idea of where the Cre is active
- Rosa26: constitutive promoter and a lacZ reporter. There also contains a stop sequence. So under normal circumstances, nothing happens, the gene is not expressed. When the cre induced, the stop codon is removed and the lacZ reporter is shown
- This gives both temporal and spatial control over the cells that are tracked.
What canbe gathered from scRNAseq analysis of embryonic human brain development?
- Detailed description of cell types
- Changes in gene expression during development
- Differences in gene expression between species
What is an organoid?
A collection of organ-specific cell types that develops from stem cells or organ progenitors and self-organisers through cell sorting and spatially restricted lineage commitment in a manner similar to in vivo
- Best model to study human embryonic development
What are 2 uses of induced pluripotent stem cells (iPSC)?
- Patient-derived iPSC’s show promise for identifying disease-causing mechanisms
- ES & iPSC’s can be genetically engineered, eg using CRISPR/Cas9 to introduce specific mutations
