Genetically modified mice

Question 1

What defines a transgenic animal?

Answer 1

• A synthetic gene or a modification of an existing gene (by addition of external DNA), is introduced during early development
• The genetic change is incorporated into somatic and germ cells and is inherited according to Mendelian laws

Question 2

What can transgenic animals be used for?

Answer 2

• basic research
• disease models
• bioreactors

Question 3

What does pronuclear injection lead to?

Answer 3

• over-expression
• misexpression
• expression of mutated proteins

Question 4

How does pronucelar injection work?

Answer 4

• DNA microinjected into fertilised mouse egg
• DNA integrates randomly into a chromosome (several copies, but only ONE integration site)
• The transgene contains its own regulatory region governing gene expression
• Chromosomal regulatory regions or DNA strucutre can disturbed the planned expression pattern

Question 5

Describe the workflow of pronuclear injection

Answer 5

1. Superovulated egg donor (fertilized eggs)
2. DNA injection 1-5ng/mL 1 pl/cell
3. Implantation in pseudopregnant female
4. pups born 3 weeks later
5. tail biopsy
6. DNA analysis: detection of transgene

Question 6

What are founder mice?

Answer 6

The pups following pronuclear injection that carry the transgene (20-30% of all pups). Each of these funders gives rise to a separate transgenic mouse line (crossed with WT, 50% of offspring are transgenic)

Question 7

What is blastocyst injection / how does it work?

Answer 7

• change a specific gene in the chromosomal DNA (not random)
• very unusual event - direct microinjection of DNA does not work efficiently
• genetic modification is done in embryonic stem cells
• genetically modified stem cells are injected into early embryo (blastocyst)

Question 8

Which kind of transgenic animals can be produced with blastocyst injection?

Answer 8

• knock-out mice
  
  • deletion of a specific gene in a whole organism
• knock-in mice
  
  • exchange one gene for another
    
    • WT changed to a totally different gene
    • WT changed to a mutated gene
• conditional knock-out mouse
  
  • inactiviation of a specific gene in a specific tissue and or at a specific time point

Question 9

How to prepare for blastocyst injection

Answer 9

• design and construct DNA targeting vector (containing antibiotic resistance gene)
• transfect ECS cells with targeting vector –> homologous recombination needed!
• grow cells with antibiotics, only cells that have integrated targeting vector survive
• pick individual colonies
• analyse the correct ESCs
• ESCs are expanded, injected to blastocysts and transferred to pseudopregnant female

Question 10

What is a chimera?

Answer 10

• A mouse that carries the ESCs in addition to its own genes
• When germline transmission, it can transmit modification to the next generation

Question 11

Workflow of blastocyst injection

Answer 11

• isolate BC
• culture ES cells
• gene targeting
• genetically modified ESCs
• inject into blastocyst
• reimplant into foster mother
• get chimeras
• cross two heterozygous chimera
• 25% of offspring will be homozygous for KO/will be KO
• mate these homozygous mice if viable and fertile

Question 12

Solutions to the problem with the background strain

Answer 12

• breed your mutant strain with the desired background strain (about 10 generations)
• make the mutation directly in the desired strain
  
  • pronuclear injection:
    
    • different strains are more or less responsive to superovulation.
    • eggs from various mouse strains differ in pronuclear size, survival rate after injection etc
  • blastocyst injection
    
    • Only ESCs from very few strains available

Question 13

What to do when the transgene is not expressed when/where expected

(surrounding DNA sequences disturb expression)

Answer 13

• use transgene with very long control regions
• insert DNA in permissive chromosomal region where expression is not disturbed

Question 14

Name three new tools for genome engineering

Answer 14

• zinc finger nucleases (ZFNs)
• transcription activator-like effector nucleases (TALENs)
• CRISPR/Cas (RNA guided cleavage)

Based on sequence specific cleavage of genomic DNA, requires NHEJ