Mouse Models for Genetics

Question 1

Examples of Nuclease Mediated Recombination

Answer 1

Zinc-finger nucleases
TALEN nucleases
CRISPR-Cas9

Question 2

Genetic Diversity of Mice

Answer 2

More diverse population = Harder to get significant results

Remember: Mice =/= Humans, Mice =/= Other Mice

Question 3

Inbred Strains

Answer 3

Backcrossed for at least 20 generations.

Used to control genetic diversity.

Question 4

Transgenic Model

Answer 4

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.

Question 5

Constructing a Transgene

Answer 5

1. Open Reading Frame of the Gene of Interest.
2. PolyA. Sequence
3. Promotor or Enhancer

Question 6

Inserting the Transgene

Answer 6

1. Pronuclear Microinjection
2. Retrovirus Mediated Gene Transfer
3. Oocytes cultured and transferred to recipient female

Question 7

Problems with Transgenes

Answer 7

“Positional Effect”
Multiple Site Insertion/Copy Number
Can be overcome by using recombinases to control the locus in which the transgene is inserted

Question 8

Gene Targeted Model

Answer 8

Animal which has an alteration in an endogenous gene. Use knock-out and knock-in modifications. Slower and more complex model.

Question 9

Embryonic Stem Cells

Answer 9

Pluripotent, isolated from inner cell mass of blastocysts, can be maintained in culture in an undifferentiated state

Question 10

Generating Gene Targeted Mice

Answer 10

1. Targeting Vector (Create a targeting vector)
2. ES Cell Targeting (Use targeting vector to target the ES)
3. ES Cell Screening (Screen ES to see if DNA is incorporated correctly)
4. Microinjection Into Blastocysts (Inject ES into a blastocyst and reimplant into the female)
5. Chimaeric Mice (This produces chimaeric mice which can be identified by their coat colour)
6. Germline Transmission (Determine if the modified ES cells have contributed to the chimaera’s germline)
7. Obtain Homozygous Animals (Breed heterozygote chimaera’s to produce to obtain homozygotic mice)

Question 11

Problems With Knock-outs

Answer 11

1. Size of Deletion
2. Genetic Background
3. Embryonic Lethality
4. Lack of Observable Phenotype
5. Distinguishing Primary and Secondary Effects

Question 12

Knock-in Mutations

Answer 12

Point mutation of a specific residue

Question 13

Conditional Knock-outs

Answer 13

Knockout at a specific developmental stage in a specific tissue.