Animal tissues and development Flashcards
Animal Cloning in Mammals
- Used unfertilised egg.
- Removed spindle to make vessel.
- Cultured udder cells which had all genetic material.
- Transferred nucleus of udder cell into empty site in vessel.
- Embryo was then cultured and implanted into a sheep.
- The lamb born was genetically identical to the nuclear donor.
Cloning taught us that…
- DNA is not lost in specialised cells during development.
- Gene expression must be regulated so that differentiation can occur.
- DNA can be reprogrammed following differentiation, to form any cell type in the body.
Considerations for choosing an animal model
- Easy to maintain?
- Easy to breed?
- Costs?
- Experimental advantages?
- Known genome sequence?
- Ethical considerations?
- Animal licence?
What makes a species good for studying genetics?
- Large number of offspring
- Short generation/gestation time
- Large array of mutants available
What makes a species good for studying embryology?
- Large number of embryos
- External development
- Robust, easy to manipulate embryos
What makes a species good for studying genomics?
- Relevance to human genome
- (gene conservation)
- Disease models
- Drug testing
Early developmental processes in the frog
- Frogs are vertebrates.
- Frogs are amphibians.
- Frogs undergo metamorphosis.
- Good model organism for experimental embryology
Poles in frogs eggs/embryos
- animal pole
- egg-rich vegetal pole.
- In some frog species the animal pole is darkly pigmented.
Cell divisions occur without an increase in size of the blastula; this means that the cells become …
… progressively smaller.
Do cells of the yolk pole or animal pole divide more rapidly?
The blastomeres (cells of the animal pole) divide more rapidly than the yolk cells (cells of the vegetal pole) and therefore become more numerous.
Where does the sperm enter
- The sperm enters somewhere at the animal pole.
- Rotation of the cortical cytoplasm then takes place revealing a grey crescent region opposite the sperm entry point.
- The dorsal lip is just below this grey crescent region.
Gastrulation in frogs
- Forms the multi-layered embryo.
- Cells move into the embryo and generate the 3 germ layers; the ectoderm, mesoderm and endoderm.
- The animal pole cells at the dorsal lip of the blastopore begin to involute
- The archenteron (primitive gut) is formed, displacing the blastocoel.
- The animal pole cells on the ventral side of the blastula meanwhile envelop the vegetal pole cells by epiboly, followed by some involution through the ventral lip of the blastopore.
- The head end (anterior end) will be formed at the leading edge of the mesoderm
Involute
Causes the future mesodermal and endodermal cells to be moved interiorly.
Neurulation
- Occurs after gastrulation and forms the closed neural tube.
- Raised neural folds appear either side of a neural groove.
- The folds move together to form a closed neural tube.
Cell divisions in frog embryos occur without an increase in size of the …
… blastula. This means that cells get progressively smaller.
Cleavage in mammals can be …, in contrast to the … cleavage of frogs.
Asynchronous
Regular
Superficial cleavage
As in drosophilia zygotes (insects)
Rapid nuclear divisions without forming separate cells.
Invagination
When cells around the outside of an embryo move inside the embryo by forming a pocket of cells into the embryo.
Involution
When cells move inside the embryo as a sheet on the inside edge.
Ingression
When you have a sheet of cells, some of which lose their connectivity and move into the embryo as single cells.
Delamination
When a whole sheet enters the embryo, like ingression.
Epiboly
Where cells spread and cover the entire early embryo.
Genes wash over the embryo in a gradient. Because of this, …
… different genes are turned on and off. This causes body plan regions within the embryo.
Forward genetics
- Start with random mutation
- Find phenotype
- Find gene
Reverse genetics
- Start with gene
- Remove gene
- Look at phenotype
Genetic screens
- Forward genetics
- Identify and study a phenotype within a mutated population
1. Generate random mutants
2. Oserve phenotype of random mutants
3. Identify the gene/s that are mutated
- Generate random mutants
Genetic screeens
- Male fish are mutagenized with chemical agent.
- Allow males and females to reproduce producing F1 (+/-) and (+/+)
- Each F1 is bred with wild-type (+/+) partner (produces F2 families)
- F2 inbred to get F3 (can now see some (-/-) phenotypes, 1/16 of them).
- Identify the gene/s that are mutated
Genetic screens
In situ hybridisation can be used – you can see the RNA product of a gene and find where it was expressed.
* Embryos fixed and mixed with RNA tag and antibodies that are labelled with an enzyme.
* Embryos are washed and the substrate reacts with the enzyme.
Genetic engineering
- Reverse genetics
- A technique used to modify gene expression within an organism
- Includes transgenesis and targeted knockouts.
Transgenesis
- Addition of a transgene
- Plasmid DNA with gene of interest and a detection marker
- The location where the gene is added within the genome is random
- Plasmid DNA is inserted into fertilised zygotes before being replanted into a host.
Targeted knockouts
- Generate a targeting vector
- Obtain embryonic stem cells
- Genetically modify ES cells
- Inject modified ES cells into blastocyte - recombinant
- Implant recombinant embryos into pseudopregnant surrogates
- Screen for chimeric mice
- Breed the chimeric mice
Generate a targeting vector
Targeted knockouts
Disrupted gene of interest means no mRNA can be produced
Obtain embryonic stem cells
Targeted knockouts
Requires undifferentiated ES cellls growing in culture
Genetically modify ES cells
Targeted knockouts
Homologous recombination can cause swapping out of host DNA for added DNA
Chimeric mice
Have a mixture of normal cells and targeted cellls.
Often coat colour markers are used.
