Mouse Models for Genetics Flashcards
Examples of Nuclease Mediated Recombination
Zinc-finger nucleases
TALEN nucleases
CRISPR-Cas9
Genetic Diversity of Mice
More diverse population = Harder to get significant results
Remember: Mice =/= Humans, Mice =/= Other Mice
Inbred Strains
Backcrossed for at least 20 generations.
Used to control genetic diversity.
Transgenic Model
Insertion of a foreign gene or DNA into a genome.
Add gene along with a specific promotor and add tags for visualisation of protein expression. Fast Technique.
Constructing a Transgene
- Open Reading Frame of the Gene of Interest.
- PolyA. Sequence
- Promotor or Enhancer
Inserting the Transgene
- Pronuclear Microinjection
- Retrovirus Mediated Gene Transfer
- Oocytes cultured and transferred to recipient female
Problems with Transgenes
“Positional Effect”
Multiple Site Insertion/Copy Number
Can be overcome by using recombinases to control the locus in which the transgene is inserted
Gene Targeted Model
Animal which has an alteration in an endogenous gene. Use knock-out and knock-in modifications. Slower and more complex model.
Embryonic Stem Cells
Pluripotent, isolated from inner cell mass of blastocysts, can be maintained in culture in an undifferentiated state
Generating Gene Targeted Mice
- Targeting Vector (Create a targeting vector)
- ES Cell Targeting (Use targeting vector to target the ES)
- ES Cell Screening (Screen ES to see if DNA is incorporated correctly)
- Microinjection Into Blastocysts (Inject ES into a blastocyst and reimplant into the female)
- Chimaeric Mice (This produces chimaeric mice which can be identified by their coat colour)
- Germline Transmission (Determine if the modified ES cells have contributed to the chimaera’s germline)
- Obtain Homozygous Animals (Breed heterozygote chimaera’s to produce to obtain homozygotic mice)
Problems With Knock-outs
- Size of Deletion
- Genetic Background
- Embryonic Lethality
- Lack of Observable Phenotype
- Distinguishing Primary and Secondary Effects
Knock-in Mutations
Point mutation of a specific residue
Conditional Knock-outs
Knockout at a specific developmental stage in a specific tissue.