Methods In Development Flashcards
(24 cards)
What is fate mapping and why is it important?
fate mapping = process of tracing the developmental outcome of specific cells or regions in an embryo
- helps us understand how cells contribute to tissues/organs during development
- insights into cell fate and lineage tracing
How are genetic markers used in fate mapping?
genetic markers (e.g. GFP and beta-galactosidase (lacZ))
- label cells in transgenic organisms
- enables continuous tracking of cells over time
- retroviruses and chimeras (e.g., chick-quail) can also trace cell lineages and developmental paths in embryos
common chemical markers used in fate mapping?
vital dyes - label surface cells for tracking tissue development
radiolabelling - labels DNA; study cell division & migration
enzymes - histological analysis
carbocyanine dyes - tracks membrane-bound cells (neuronal studies)
fluorescent dextrans - live imaging of cells
What is photoconversion and how does it aid in fate mapping?
photoconversion = involves a fluorescent protein that changes colour when exposed to specific light wavelengths
- allows precise tracking of labelled cells over time
- can study cell lineage and migration
How do chimeras help in fate mapping studies?
chimeras = organisms made from cells of two embryos, often from different species (e.g., chick-quail chimeras)
- can trace how cells from one organism contribute to development in another
- insights into cell migration and developmental interactions
What are transgenics? How do transgenics contribute to fate mapping?
transgenics = organisms genetically modified to stably express foreign DNA (e.g. GFP reporter genes) - allows long-term, inheritable labelling of specific cells/tissues
- visual tracking of cell migration, development contributions, differentiation
- high-resolution live imaging
What is grafting and how is it used in fate mapping?
grafting = transplanting a piece of tissue from one embryo to another
- orthotopic (same location) or heterotopic (different location)
- helps study how tissues behave when placed in different environments & tracks their fate in development
What are the classical approaches in descriptive embryology?
- light microscopy (gross morphology)
- electron microscopy (ultrastructural details
- histology (tissue organization)
- fluorescent labelling (live tracking of cells)
provide insights into normal embryonic development and tissue differentiation
pros of fate mapping?
- continuous tracking of individual cells or tissue regions over time (e.g., GFP, photoconversion)
- versatile across species and stages of development
- allows specific lineage tracing with genetic markers
cons of fate mapping?
- limited resolution (e.g., dye labelling) = not always high-resolution enough to track single cells in complex tissues
- short-term vs. long-term tracking = some techniques (vital dyes) are useful only for short-term studies; others (transgenics) better for long-term studies but are more technically challenging
- potential off-target effects with genetic manipulation
What is forward genetics and how is it used to study development?
forward genetics = creating random mutations (by chemicals or radiation) and screening embryos for phenotypic changes
- mutants with abnormal development are isolated = mutated gene identified
- helps discover unknown genes involved in development
What is reverse genetics and how does it help study developmental genes?
reverse genetics = known gene mutated/deleted deliberately; resulting phenotype analysed
- directly tests a gene’s specific role in development
What are mutagenesis screens and what do they reveal?
mutagenesis screens (forward genetics) = induce random mutations and screening embryos for developmental defects
- reveal genes that are essential for normal embryogenesis
how are ZFNs and TALENs used for targeted genome editing in reverse genetics?
consist of a DNA binding domain & cutting domain
- DNA binding domains bind to specific DNA sequences
- cutting domains cut DNA
- cells tries to repair DNA itself but makes mistakes - insertions/mutations
How does CRISPR-Cas9 work in gene editing?
- guide RNA leads Cas9 enzyme to a specific DNA sequence - makes a cut
- cell repairs the break via NHEJ (non-homologous end joining; error-prone, knockouts) OR HDR (homology-directed repair; precise edits if a template is given)
- type of repair allows targeted gene disruption/modification
how do forward and reverse genetics fundamentally differ?
forward genetics - start with phenotype, find the gene
reverse genetics - start with known gene, create mutation/deletion, observe phenotype
pros and cons of CRISPR-Cas9?
pros:
- fast, cheap, efficient
- targets many genes at once
cons:
- off-target effects with unwanted cuts
- not all cells are edited; mosaicism
- HDR/ homology-directed repair is less efficient despite more precise edits
What is subtractive hybridisation and what is it used for?
subtractive hybridisation = compares two mRNA samples (e.g. normal vs mutant) to remove common genes and isolate genes differentially expressed
helps identify genes specific to a tissue/condition
how does subtractive hybridisation work?
- isolate mRNA from two samples.
- make cDNA and tag one sample (e.g., biotin).
- hybridise together — shared sequences bind.
- remove common sequences (e.g., via streptavidin).
- remaining unbound cDNA = differentially expressed genes.
How can we study if genes function in the same molecular pathway?
- analysing gene expression patterns
- performing misexpression studies (gain/loss of function)
- conducting genetic epistasis tests
helps infer the order and interaction of genes
what is genetic epistasis? how is it used? how is genetic epistasis interpreted in repressive pathways?
genetic epistasis = studies the relationship between two genes by creating single and double mutants and analysing phenotypes
helps determine gene order in regulatory pathways
repressive pathways - if two genes repress each other, double mutants often show the phenotype of the gene acting last - comparing opposite phenotypes in single vs double mutants clarifies regulatory order
What are misexpression studies? How do misexpression studies help understand gene function? (gain and loss of function)
misexpression = altering expression of a gene (level/ time/ location) - observe its effect on development & other genes’ expression
gain of function: force expression of a gene in a new place/time - reveals sufficiency (gene is enough on its own to cause a specific developmental process)
loss of function: knockout/ knockdown a gene - shows necessity for a process
How can misexpression be achieved experimentally?
retroviruses
injected mRNA
transgenic animals carrying chimeric genes
dominant-negative vs dominant-positive construct/ mutant receptor for studying gene expression?
dominant-negative = removes the intracellular signalling domain of a receptor - receptor still binds the ligand but blocks signal transduction; sequestering the ligand and causing a loss of function
dominant-positive = designed to signal even without a ligand; constant pathway activation leading to gain of function phenotypes
