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Flashcards in Genetically modified mice Deck (14):
1

What defines a transgenic animal?

  • 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

2

What can transgenic animals be used for?

  • basic research
  • disease models
  • bioreactors

3

What does pronuclear injection lead to?

  • over-expression
  • misexpression
  • expression of mutated proteins

4

How does pronucelar injection work?

  • 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

5

Describe the workflow of pronuclear injection

  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

6

What are founder mice?

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)

7

What is blastocyst injection / how does it work?

  • 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)

8

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

  • 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

9

How to prepare for blastocyst injection

  • 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

10

What is a chimera?

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

11

Workflow of blastocyst injection

  • 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

12

Solutions to the problem with the background strain

  • 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

13

What to do when the transgene is not expressed when/where expected
(surrounding DNA sequences disturb expression)

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

14

Name three new tools for genome engineering

  • 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