Introduction to the generation and use of genetically modified mice

Question 1

How can we modify the mouse genome?

Answer 1

• Point mutations
• Insertions
• Deletions
• Inversions
• Duplications
• Translocations
• Expansions
• Aneuploidy
• Copy number variations

Question 2

What are modifications within the mouse genome used for?

Answer 2

• Studying gene function, protein function
• Replicating a human genetic defect in the mouse
• Creating a model of human genetic disease
• Humanisation as a means of modelling disease

Question 3

What are the constraints of using mice as a model of human disease?

Answer 3

• Mice are not human
• Genetic background makes a difference
• Gene dosage can have different effects between human and mouse.
• Complex phenotypes are difficult to replicate
  
  • eg neurobehavioral defects

Question 4

How is the DNA modified to form the mRNA molecule?

Answer 4

-introns are removed from the hnRNA and a 5’ cap and a 3’ polyA tail is added to give mRNA

Question 5

What is transgenesis?

Answer 5

Transgenesis is the stable insertion of exogenous DNA into host cell’s chromosomal DNA (does not include transient transfection)

Question 6

What is the earliest transgenics based on and what is this probably due to?

Answer 6

• Earliest transgenics based on the fact that exogenous DNA added into a nucleus (or even into cytoplasm) sometimes becomes integrated into the cell’s own chromosomal DNA
  - probably due to the action of DNA repair enzymes

Question 7

What does exogenous DNA being same to endogenous DNA lead to in transgenesis?

Answer 7

• Exogenous DNA with the same sequence as endogenous DNA can lead to exchange
  - This is known as homologous recombination

Question 8

What do genome engineering techniques in transgenesis use?

Answer 8

• Genome engineering techniques used modified nucleases to cut, or nick genomic DNA
• endogenous DNA repair enzymes cause mutations or can be directed to insert novel DNA sequences

Question 9

Why is embryo manipulation important for transgenesis and how is this done?

Answer 9

Embryo manipulation is important for transgenesis:

• Usually we wish to produce a permanent, stable, genetic model to pass onto offspring
• So, we need to generate a germ line genetic modification
• Thus we need to manipulate germ cells or the very early embryo

Question 10

What is transgenic research further dependent on?

Answer 10

• The availability of pluripotent embryonic stem cells
• The harnessing of DNA homologous recombination
• shRNAi, recombinases, nucleases, and other molecular technologies

Question 11

Random transgenesis

Answer 11

• DNA microinjection into zygotic pronucleus
• Relatively quick and easy
• Transient assays possible

Question 12

Targeted 1 transgenesis

Answer 12

• Homologous recombination in embryonic stem cells
• Relatively slow and difficult
• Very powerful and flexible

Question 13

Targeted 2 transgenesis

Answer 13

• Genome engineering techniques in embryonic stem cells
• Ease and speed depend on technique used
• Possibility of off target effects

Question 14

Steps in the preimplantation development

Answer 14

• A fertilised mouse egg(zygote), the male and female pro nucleus don’t immediately fuse
• Male pro-nucleus is usually larger than the female pro-nucleus
• Male and female pro nucleus fuse and mitotic division resumes
• The embryo is contained within a protein coat known as the zona pellucida
• At about the 32 cell division, we get formation of 2 distinct type of cells
  
  • Trophectoderm
  • Inner cell mass
• Derive all the cells of the embryo