Techniques 1 Flashcards
(32 cards)
Classical methods
Morphological descriptions
Histology
Cell ablation
Study of mutants
Morphological descriptions
Isolate embryos at different stages and describe their morphology
Histology
- isolate embryo
- fix using fixative e.g. paraformaldehyde
- dehydrate
- embed in wax
- section + take onto slides
- stain + observe tissue histology under microscope
Fixation
Kills tissue to prevent postmortem decay
e. g. using paraformaldehyde
- > cross-links proteins
Cell ablation
Ablation of specific cellular structures in embryo + allow it to develop
- study regeneration, development + compensation
Use lasers or toxins to kill cells
Study of mutants
e.g. Taplid chick
Taplid3 = chicken mutant w/ abnormal limb patterning + malformations in regions of embryo known to depend on Hedgehog signalling
Study of mutants
e.g. Mouse piebald mutation
Broad white patches on belly + head
Pigment called melanocytes in mouse embryos have mutations in c-kit gene
Lineage tracing
Identify all progeny of a single cell
Essential for studying stem cell properties
Help to understand tissue development, homeostasis + disease
Tissue homeostasis
Balance between cell division + cell differentiation
Fate mapping + lineage tracing of embryos
> Grafting
Vital dye marking
Fluorescent dye + radioactive labelling
Genetic marking + grafting
Tissue grafting
e.g. Spemann + Mangold
Graft a tissue from 1 embryo to another
-> graft of organiser led to conjoined twin embryos
.:. embryo’s cells are not committed to a certain fate
Naturally occurring genetic markers as lineage tracers
e.g. quail and chick cells
Cells from quail embryo grafted into chick embryo
- can determine the cell type from their heterochromatin of their nuclei
quail cells have a large nucleolus
Methods for identifying genes regulating development
> Homology searching
Cloning of cDNA and screening of libraries
Screening + sequencing of expressed sequence tags
Mapping + identifying genes responsible for a mutant phenotype
Methods to detect gene expression
- mRNA
- northern blotting
- RT-PCR
- microarrays
- in istu hybridisation - Protein
- Immunostaining
- Western blotting
Molecular hybridisation
Southern blot (DNA-DNA) Northern blot (DNA-RNA) Western blot (RNA-RNA)
Southern blot
- Restricted DNA on gel
- Denature + transfer to filter
- Hybridise with probe
= labelled DNA
Northern blot
1. RNA on gel (separated by gel electrophoresis) 2. Transfer to filter 3. Hybridise with probe = labelled DNA
Western blot
- Protein on gel
- Transfer to filter
- React with probe
= antibody
Shows which proteins are expressed
Analysis of gene expression
> Northern blotting > PCR > In situ hybridisation a) sections b) whole mounts > microarray analysis
Expression of RNA
> Quantitative assays - in situ hybridisation > Qualitative + tissue specific assays > Large scale assays - micro assays
RT-PCR
= reverse transcriptase - polymerase chain reaction
- cDNA synthesised by reverse transcriptase of mRNA
- RNase removes mRNA
- PCR - using primers specifically binding to sequence of interest
- visualisation on agarose gel (after staining DNA)
Microarray
- isolate mRNA from samples
- RT + fluorescent labelling (diff colours for diff samples)
- > cDNA - Hybridisation to microarray
4. Chip contains sequences representing diff genes On chip: Green = mRNA only present in sample 1 Red= mRNA only in s2 Orange = mRNA in both Black = mRNA in neither
In situ hybridisation
This method either:
1. localises mRNA within cytoplasm
or 2. DNA within chromosomes of nucleus
Hybridise sequence of interest to a complementary strand of a nucleotide probe
- Labelled DNA or RNA probe (e.g. with digoxigenin) complementary to target mRNA
- Labelled antibody (e.g. anti-digoxigenin) binds to cDNA-probe complex
Methods to manipulate gene expression
> Overexpression/ ectopic expression of genes:
- transgene expression systems
> Reduced gene expression
- dominant negative proteins
- gene targeting by homologous recombination
- RNAi
- morpholinos
- mutagenesis screens
