List the Progressive stages in the development of embryos:
- Cleavage divisions
- Pattern formation:
- body axes
- germ layer specification - Morphogenesis:
- gastrulation/neurulation - Cell differentiation
- Growth
Describe the different approaches to studying developmental biology:
- Anatomical approach:
- What parts of the embryo form different organs?
- What are the changes in tissues in birth defects? - Experimental approach:
- How do molecules or processes cause visible changes in embryos?
- How do cells order themselves into tissues and organs? - Genetic approach:
- How do genes control development?
Define a fate map and given an example:
- Marking cells in the early embryo call allow for the NORMAL fate of these cells to be followed and mapped
- If each cell in the early embryo is marked, a fate map can be formed
- The cells can be marked by:
1. Tracer/fluorescent markers
2. Genetic markers
3. Cellular/species specific markers
Describe techniques used in experimental embryology:
- Defect experiments:
- A portion of the embryo is destroyed and the impact on function is observed
e. g. remove AER and observe effect - Transplantation/Recombination experiments:
- One part of an embryo is removed and replaced with a part from a different site in the same embryo - Isolation experiment:
- Removing part of an embryo and observing it in vitro
Explain how cell fate is progressively obtained:
- Cell fate refers to what cells will normally develop into
- Cells are first specified, then determined
- When cells are determined: tissue is committed and fate cannot be reversed even when placed in a different region of the embryo (can be tested with a transplantation experiment)
What is a forward genetics approach and give an example:
- Forward genetics goes from phenotype to genotype
- It involves finding the genes responsible for a phenotype
- Spontaneous mutations/deliberate mutagenesis -> developmental abnormalities -> identification of the mutated gene
- Approaches include:
1. spontaneous mutants- look at animals with a specific defect and try and find the gene that is mutated in all of them
2. Deliberate mutagenesis (animals are given a mutagen such as ENU and the phenotype is screened for after) and genetic screens
What is a reverse genetics approach and give an example:
- Reverse genetics starts with genotype and goes to phenotype
- It involves manipulating a specific gene and looking at the associated phenotype
- Identify developmentally important gene -> introduce specific mutations -> analysis of developmental consequences
Approaches include:
1. Knockouts
2. Transgenics
Define transgenic and knockout:
Transgenic:
- Changing the genetic constitution of an organism by genetic engineering e.g. inserting a gene
Knockout:
- The complete inactivation of a particular gene in an organism
List the progressive stages of producing a transgenic mouse model:
- If you want the gene expressed in particular tissues, you use tissue specific promoters to drive the expression of a gene
- You combine you tissue specific promoter, your gene and a poly-A-tail
- The gene construct is injected into the pronucleus of a male fertilised egg at the 1 cell stage (pronuclear injection)
- The zygote is transferred to a host mouse
- The gene can be tested for genetically or it can be linked to a marker gene e.g. GFP, so GFP will be expressed if that transgene is
Give an overview of how a KO mouse is made:
- The DNA construct is introduced into embryonic stem cells (these are derived from the ICM of a blastocyst)
- Colonies of ES cells containing the desired modification are screened for
- ES cells are injected into a host blastocyst and then transplanted into a surrogate
- Chimeric mice are born and can be bred to produce a knockout animal
Give an overview of how a KO mouse is made:
- The DNA construct is introduced into embryonic stem cells (these are derived from the ICM of a blastocyst):
- This involves creating a targeting DNA construct that contains a drug-resistance marker
- The targeting DNA is introduced using electroporation - Colonies of ES cells containing the desired modification are screened for
- As the gene targeting DNA contains a drug-resistance gene the ES cells can be screened for using that drug - ES cells are injected into a host blastocyst and then transplanted into a surrogate uterus
- The altered ES cells will be from a mouse of one colour e.g. white, and will be injected into the blastocyst of a different colour e.g. brown - Chimeric mice are born and can be bred to produce a knockout animal
- The chimeric mice will be a mixture of ES cells and host blastocyst cells
- The chimeric mice can be bred with a wild-type mouse to produce mice that are heterozygous for the introduced mutation
- The heterozygous mice can be bred to produce a homozygois knockout
- If the knockout is embryonic lethal no homozygous mutants will be produced and the mendellian ratio will be 2:1