Generation and Use of Transgenic Animals

Question 1

Why do we use genetically modified animals (GMA)?

Answer 1

• Identify range of genetic changes that contribute to human disease, mapping genome
• Can model these genetic changes from humans

Question 2

What mechanisms are used to create these genomic mutations?

Answer 2

• X-rays (double stranded breaks but humans usually single base mutations)
• Ethyl nitrosourea (ENU)
• Ethyl methane sulphonate (both ethyl = single point mutations = mutate sperm)

Question 3

How is ENU used to generate mutations?

Answer 3

ENY fed to male mice = mutate sperm = 87% AT, 13% GC mutations = mutant proteins (64% missense, 26% splice site, 10% nonsense) = mutant phenotypes = human disease models

Question 4

How is microinjection used to generate transgenic mice?

Answer 4

1. Fertilized undivided eggs are isolated from female mice (single cell embryo before male and female nuclei have fused)
2. M and F pronucleus is visible under microscope
3. Holding-capillary used to grip the egg under vacuum
4. DNA is introduced into the egg male pronuclei by microinjection
5. Microinjection (Pro-nuclear mediated transgene introduction) - small DNA injection volume (~3 picolitres) performed via a very fine glass micropipette that’s able to penetrate the cell membrane of the fertilized ovum without damage
6. Usually integrates tandemly (too many copies)

Question 5

What are transgenics?

Answer 5

The intentional introduction of a foreign gene or genetic construct into the genome of a target animal (1st mouse - 1982: metallothionein-1 gene promotor = big mice)

Question 6

How do you isolate embryonic stem cells to make exact changes in a gene?

Answer 6

1. Blastocysts = fertilized early developing embryoes that havent attached to the uterus, totipotent
2. Blastocysts are isolated from an early mouse embryo
3. Blastocysts are cultured on feeder embryonic fibroblasts
4. Blastocyst attaches to feeder layer and the ICM begins to grow
5. ICM expands to give embryonic stem (ES) cells
6. Individual ES cell lines tested for their ability to make chimeric mice and can be derived in vitro to give pluripotent cells
   - Can take cells from epiblast of developing blastocyst = pluripotent cells (won’t make uterus)

Question 7

How do you create a congenic line?

Answer 7

• Can link gene in ES cell to colouration so when you transfer the ES cell you know white mice have the gene
• Backcross chimera mice (w/ ES) with C57BL/6 for 10 generations until you end up with a fragment on the genome aorund the gene you integrated
• You can get exact genes but takes 1-2 years
• Also get other genes integrating from chimera = weird phenotypes

Question 8

How do you edit genomes with sequence-specific nucleases (CRISPR)?

Answer 8

• Tail ends and sing finger nucleases use protein sequences to identify nucleotide sequences = more cumbersome to produce than CRISPR Cas 9 (uses RNA molecules)
• CRISPR hybridizes to region you’re interested and cuts the genome in a specific region, making double stranded breaks
• Can get two results:
  -> Non-homologous end joining = cell takes break, chews off ~30 nucleotides and sticks it back together = knockout of gene, needs to be in wrong frame to create knockout
  -> Broken segments search for sequence w/ homology = damaged strands search for another copy of gene and repair by homologous recombination. Can also give donor DNA w/ homologous sequence either side of the damage site = integrate into genome = engineer many changes

Question 9

What genome modifications are generated by the Trinity transgenic facility?

Answer 9

• Introduce stop codons/point mutations, integrated reporters/inducible regulators, introduce humanized genes