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Flashcards in Week 9 Deck (11)
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1
Q

List the Progressive stages in the development of embryos:

A
  1. Cleavage divisions
  2. Pattern formation:
    - body axes
    - germ layer specification
  3. Morphogenesis:
    - gastrulation/neurulation
  4. Cell differentiation
  5. Growth
2
Q

Describe the different approaches to studying developmental biology:

A
  1. Anatomical approach:
    - What parts of the embryo form different organs?
    - What are the changes in tissues in birth defects?
  2. Experimental approach:
    - How do molecules or processes cause visible changes in embryos?
    - How do cells order themselves into tissues and organs?
  3. Genetic approach:
    - How do genes control development?
3
Q

Define a fate map and given an example:

A
  • 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
4
Q

Describe techniques used in experimental embryology:

A
  1. Defect experiments:
    - A portion of the embryo is destroyed and the impact on function is observed
    e. g. remove AER and observe effect
  2. Transplantation/Recombination experiments:
    - One part of an embryo is removed and replaced with a part from a different site in the same embryo
  3. Isolation experiment:
    - Removing part of an embryo and observing it in vitro
5
Q

Explain how cell fate is progressively obtained:

A
  • 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)
6
Q

What is a forward genetics approach and give an example:

A
  • 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
7
Q

What is a reverse genetics approach and give an example:

A
  • 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
8
Q

Define transgenic and knockout:

A

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

9
Q

List the progressive stages of producing a transgenic mouse model:

A
  1. If you want the gene expressed in particular tissues, you use tissue specific promoters to drive the expression of a gene
  2. You combine you tissue specific promoter, your gene and a poly-A-tail
  3. The gene construct is injected into the pronucleus of a male fertilised egg at the 1 cell stage (pronuclear injection)
  4. The zygote is transferred to a host mouse
  5. 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
10
Q

Give an overview of how a KO mouse is made:

A
  1. The DNA construct is introduced into embryonic stem cells (these are derived from the ICM of a blastocyst)
  2. Colonies of ES cells containing the desired modification are screened for
  3. ES cells are injected into a host blastocyst and then transplanted into a surrogate
  4. Chimeric mice are born and can be bred to produce a knockout animal
11
Q

Give an overview of how a KO mouse is made:

A
  1. 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
  2. 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
  3. 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
  4. 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