L2 + L3 - Basic Principles and Techniques Flashcards
What is pattern formation?
Process by which cells are organised in space and time to produce a well-ordered structure
Establishment of body plan and the main body axes
Cell has positional information – coordinates for each cell
What is morphogenesis?
Cell and tissue movement and changes in cell behaviour that give developing organ its 3D shape
What does morphogenesis involve?
Cell adhesion
Cell migration
Cell death – e.g. formation of digits
Cell shape
What is cell differentiation?
Process by which cells become different from each other and acquire specialised properties
Governed by changes in gene expression which dictate the repertoire of protein synthesised
Progressive restriction of pluripotency
The step wise process of cell differentiation?
Cell specification
Determination
Differentiation
Maturation
What is growth?
An increase in mass or size
It is a continuous process
Growth rate varies depending on age and organ
Cell proliferation, cell enlargement, accretion
What are the 4 ways development is studies?
Embryology – observed biology and experimental manipulation
Developmental biology – study of genes and proteins
Animal models
Genetics
What are the two opposing theories of development?
Funnel model
Hourglass model
What is the funnel model?
Haeckel
Diversity occurs at later stages
Later proved to be wrong
What is the hourglass model?
Von Baer
Early stages are very similar
Intermediate stages very similar between different animals
How to find out where and when the gene is expressed in the embryo?
In situ hybridisation – spatial expression Northern blot – spatial expression RT-PCR Micro-array Reporter lines – spatial expression
How to find out if the protein is expressed with the same timing that the gene?
Western blot
Immunohistochemistry
How to find out if the gene/protein is essential for development?
Gain of function
Loss of function
How to find out how the gene is regulated?
Embryology – tissue manipulation
Manipulating signalling pathways – drugs, transfection, electroporation, genetics
How to find out what the tissues/organs derived from the cells that express this gene?
Embryology – chick chimera, labelling with dye
Genetics – labelling with retrovirus or GFP
Study of gene expression is used to?
Establish where and when a gene is expressed
How do you study gene expression?
In site hybridisation
Reporter lines
High throughput analyses – microarray, RNAseq
In situ hybridisation
- Get reaction compound
- Incorporate a probe (anti-DIG-AlkPhos) into the compound
- Probe enters every cell and form a hybrid wherever there is mRNA
- Use an antibody this is coupled to the probe to identify where probe is bound to mRNA
Reporter lines
Reveals where a gene is expressed
Expression of the gene sequence relies on the expression of regulatory enhancer sequences
Can replace gene sequence with reporter gene
Place regulatory sequence and reporter gene into animal model
Creates a transgene which expresses GFP
What do microarrays do?
Try to compare the expression profile of all the genes in the genome between two cell populations
Microarrays method
- Start with two samples and isolate the transcripts
- Reverse transcribe transcripts to create cDNA
- Each cDNA labelled with a unique fluorescent dye
- Hybridise to an array
- Each spot on the grid has a sequence for 1 gene in the genome - The probe forms a hybrid with the cDNA on the array forming a stable complex
- Plate readers and software read the output of the images produced
RNAseq
Sequencing step allows short fragments to be sequenced of each cDNA
Use software to determine which genes are expressed
Can use if only a single cell is available
Study of proteins is used to?
Investigate the distribution of proteins
How do you study proteins?
Immuno-detection – immune-histochemistry, immune-fluorescence
Fusion protein construct
Immunofluorescence
Need prior knowledge of protein
Need a specific antibody that can recognise the protein
Need a primary antibody – specific to protein of interest
Immunofluorescence method
- Take section of tissue of interest and incubate it with primary antibody
- To reveal where primary antibody has bound to protein use a secondary antibody (IGg coupled to fluorescent tag) which binds to primary antibody
Fusion protein construct
Fusion protein - tagged with GFP
Protein expressed in tissue in which it is normally expressed – can now visualise it
Fusion protein construct method
- Start with a DNA fragment that contains the regulatory sequence of gene of interest
- Leave gene of interest in place
- Insert the DNA sequence encoding GFP in frame with the DNA sequence of protein of interest
- Insert this in 5’ or in 3’
- Want to create a single transcript that contains mRNA for GFP and mRNA for protein of interest
- This is translated creating a single protein with GFP fused to it
What are developmental genetics used for?
Determine the function of genes
How do you study developmental genetics?
Gain of function – transgenesis
Loss of function – forward and reverse genetics
Forward genetics conditions
Animal models – c.elegans, drosophila, zebrafish, mouse
Agent – chemical mutagen
Selection – based on phenotype of interest
ENU – N-ethyl N-nitrosourea
Forward genetics - positional cloning of mutated gene
Animals randomly mutated Cross mutated and wildtype - Mixed offspring Cross offspring to wild type - If wildtype – wildtype offspring - If heterozygous for mutation – 50% offspring will carry mutation Cross offspring together - 25% homozygous for mutation - 25% wildtype - 50% heterozygous for mutation
Reverse genetics method
- Homologous recombination
- ES cell transfection
- ES cell selection
- ES cell injection into blastocyst
- Implantation into mouse
- Selection of chimeric mouse
- Breed
Reverse genetics - breeding options
If successful integration of mutation into germline – mouse with mutation in every gene
Conventional knockout mouse
Conditional knockout mouse – floxed allele
What does reverse genetics allow you to test for?
Allows you to test if a gene is essential for the development of the embryo
What is the problem with reverse genetics?
If you have a gene coding for a protein with an essential function very early on in embryogenesis, you will never get to the point you can analyse various tissues – embryos die very early on
Alternative strategy to reverse genetics?
Generate conditional knockout
Allows you to bypass the requirement for a specific gene in early and embryogenesis and study its function in later stages of development
What do conditional knouts give you the ability to determine?
In a spatial manor where the knockout will occur
In a temporal manor when the knockout will be carried out
Conditional knockout method
- Flank gene of interest with LOX-P sites which are recognised by Cre recombinase
- Insert into ES cells
- Generate a mouse which contains LOX-P sites on either side of gene of interest
- Cross this mouse with mouse that expresses Cre recombinase gene
- Cre recombinase and tissue specific promotor
- Cre recombinase and a promoter controlled in a temporal manor
What is tissue manipulation used for?
Demonstrate inductive function
How do you study tissue manipulation?
Tissue ablation
Tissue graft
Tissue transplantation
Bead/cell implantation
Tissue manipulation in chick
John Saunders identified area of limb that had organiser function
- Identified using a grafting experiment
- Posterior to anterior transplantation - duplicated the chick foot
What is observational biology used for?
Fate maps and lineage analysis
How do you study observational biology?
Cell/tissue transplantation - Spemann and Mangold
Cell/tissue labeling with dye
Cell/tissue labeling genetically - electroporation, GFP transgenic lines, brainbow mice and zebrabow
Chick and quail studies
Quail and chick embryos are very similar
There are antibodies that recognise proteins at the surface of the nucleus of the quail embryo but not the chick embryo
Chick and quail studies method
- Tissues from quail transplanted early in development into chick host
- Carry out immunohistochemistry later on to identify location of antibody against quail cells
- Quail tissue will now cover a very specific area
Cant get single cell resolution
Brain bow technique in mouse and zebrafish method
- Create a transgenic reporter
- Introduce various genes encoding different fluorescent dyes
- Each constructs are framed by distinct LOX-P sites
- Important as can only have one type of incision - Introduce this into the animal model
- Once you cross the animal with Cre recombinase you have a of having a wide range of combination of colours in each of the cells were recombination has taken place
- Because of the unique colour of each colour we can follow the future fate of the cells
