Please enable JavaScript.

Coggle requires JavaScript to display documents.

DEVELOPMENTAL GENETICS - Coggle Diagram

- - - - first step is to generate the mutants
        
        a male animal (eg. fly or fish) is exposed to a mutagen which causes changes in its DNA, including changes in the DNA of its sperm
        
        this is then mated to a wild type animal to produce individual F1 animals
        
        some of these animals will have a mutation in there DNA and some will be normal
        
        individuals that have inherited the mutation will appear mutated if the gene is dominant
        
        at this stage it is usually not known which individuals do/do not have the mutation due to the nature of recessive mutations
        
        further round of breeding needed in this circumstance
        
        F1 animals need to be mated to wild type animals to produce families of F2 animals (again, it is usually not known at this stage which individuals do/do not have the mutation
        
        each F2 animal is then crossed to another F2 animal within its 'family' to produce an F3 generation
        
        at this stage it is possible to identify the families which contain a mutation
        
        only 1/16 of the animals from this cross will be homozygous mutant and therefore appear mutated
        
        1 more item...
      - second step is to identify the gene which is mutated to cause the phenotype
        
        the gene is often given the same name as the mutant
        
        eg. the gene responsible for the Ultrabithorax mutation is called ultrabithorax
        
        the 7 homeotic genes, which are also called Hox genes, were all found to encode for homeobox transcription factors
        
        all found close together in the genome
        
        homeobox transcription factors can activate expression of the genes required to produce certain cell types
        
        head
        
        labial (lab)
        
        gives antennae (wild type and mutant)
        
        deformed (Dfd)
        
        give antennae (wild types and mutant)
        
        thorax
        
        sex combs reduced (SCR)
        
        gives a pair of legs (wildtype and mutant
        
        antennapedia (Antp)
        
        gives a pair of legs and wings (wildtype and mutant)
        
        Antennapedia gain-of-function mutants
        
        lb/Dfd and Antp lose antennae and gain an extra pair of legs
        
        ultrabithorax (Ubx)
        
        Ubx and Antp gives a pair of legs and halteres (wildtype)
        
        Antp (no Ubx) gives a pair of legs and extra pair of wings - lose halteres
        
        abdomen
        
        abdominal A (abdA)
        
        Abdominal B (AbdB)
      - third step is to identify where the gene is expressed
        
        in situ hybridisation
      - a technique used to identify and study an interesting phenotype within a mutated population
    - - short generation time so crosses can be carried out quickly
      - simple genome so its easier to eventually get a mutation in every gene - 13,000 genes in 4 chromosomes
      - small and cheap to maintain and its easy to see a developmental phenotype
      - do not develop inside mother
- - - - similar homeotic transcription factor codes operate in mammals
  - - - eg. thoracic segment 3 has Ubx and Antp and gives rise to a pair of legs and halters
      - by working out the changes in the code in each region, we can work out why the changes in structures are brought about
  - - - region of the larvae which express certain Hox genes will produce certain structures in the adult fly
      - allows us to produce a transcription factor code which shows which structures are produced by which transcription factors
- - - - gene of interest is added
      - plasmid DNA coding for a gene of interest is injected into a fertilised mouse embryo
      - in some embryos this will be incorporated into the genomic DNA of the embryo
      - embryos that incorporate the plasmid into their genomic DNA will be transgenic when they are born
      - permanent genetic change (unlike RNA injection into Xenopus where the RNA is gradually degraded
      - Fertilised zygotes are removed from a female mouse and incubated in a dish
        
        to inject plasmid DNA into the zygotes, first they must be held still using a holding pipette
        
        a very fine injection pipette containing the DNA is then inserted into one of the pronuclei (this is usually always the male pronuclei which is the larger of the two)
        
        when the solution containing the DNA is expelled the pronuclei swells slightly
        
        the zygotes are then transferred into the oviduct of a pseudo pregnant recipient ( a female mouse that is in the correct stage of the oestrus cycle and has mated with a non-fertile male the previous night
        
        the DNA can integrate into the genome in a random location (or in most cases not at all)
        
        1 more item...
    - - gene of interest is removed
      - widely used in biomedical research
      - mouse embryonic stem (ES) cells are used because they can be modified in culture and then put back into a mouse where they will give rise to all the tissues of a mouse
      - ES cells can be modified by utilising:
        
        targeting vector
        
        CRISPR-Cas9
      - TARGETING VECTOR
        
        Homologous arms (5' and 3') flanking the gene of interest (the sequence of the gene to be removed)
        
        a positive selectable marker (such as a neomycin resistance gene) which is put in the middle of the sequence of the gene of interest
        
        negative selectable marker (such as thymidine kinase gene) which is toxic to cells
        
        reporter gene (such as beta-galactosidase gene) to visualise which cells would normally be expressing your gene of interest
        
        is added to ES cells
        
        in a small % of these cells homologous recombination will occur
        
        will insert the neomycin resistance gene into the middle of the gene of interest and disrupt the function of that gene
        
        because targeted cells will have neomycin resistance, if neomycin is added then non-target cells will be killed and the remaining cells must be the targeted ones
        
        although these cells are targeting they may not be correctly targeted and there are further processes to select for the correctly target cells
        
        2 more items...
        
        process is successful only rarely so there must be mechanisms to select for these rare events
        
        the targeted ES cells can then be transplanted into host embryos (blastocysts)
        
        the host embryos are then placed into a foster mother - into the uterine horns of the mouse rather than the oviduct
        
        the mice born will be a mixture of targeted cells (from the targeted ES cells) and non-targeted cells (from the host embryo)
        
        chimera
        
        needs to be mated with a wildtype mouse
        
        offspring will contain either targeted cells or non-targeted cells so none of the offspring will be chimeric
        
        1 more item...
        
        piece of DNA must be disrupted with foreign DNA to stop transcription - clump of stem cells must be desecrated and lgmDNA can be absorbed and swaps
      - eg. Pdx1 knockout
        
        Pdx1 is a transcription factor expressed in the pancreas
        
        when it is knocked-out pancreas formation is lost
        
        now thousands of mouse knockouts and they are used to study many diseases
        
        cystic fibrosis
        
        cancer
        
        heart disease
    - - mutants start with a phenotype and then the gene is found (forward genetics)
      - knockouts start with the gene under observation being knocked out, followed by looking to see if there is a change in phenotype (reverse genetics)
    - - larger genome
      - adults bigger
      - longer generation time
      - very expensive
    - - embryos snaller
      - development inside mother