Genetically modified animal models

Question 1

What is a genetically engineered mouse?

Answer 1

A mouse where a gene is in- or over- activated, by replacing or disrupting it with an artificial piece of DNA
The loss or gain of gene activity (knock-out/knock-in) often causes changes in a mouse’s phenotype

Question 2

What use is there for genetically modified mice?

Answer 2

Test specific functions of particular genes in vivo
Study the role of genes in embryonic development, aging, obesity, diseases (cancer, allergy, diabetes…), drug development, etc.

Question 3

What is the standard transgenic approach (how to do it)?

Answer 3

Several copies of the transgene DNA is microinjected into pronucleus of fertilised murine oocyte
Injected oocytes are transferred in recipient mouse
Offspring are screened for the transgene by DNA analysis
=> 10-20% of progeny carry transgene

Question 4

What is gene-targeted transgenic approach?

Answer 4

One copy of the transgene DNA is introduced into embryonic stem cells (electroporation)
Drug selection is used and surviving colonies are screened for the transgene
Cells are microinjected into mouse blastocyst
Blastocyst is transferred to recipient mouse
Chimaeric offsprings are identified and mated to test for germline transmission of the transgene
=> 25-75% of progeny carry transgene
=> goal is to aim a precise location in the genome to study

Question 5

How to create a knock-out mouse?

Answer 5

1. Prepare gene-targeting construct
2. Transfect embryonic cells (electroporation) => select for transfectants (drug selection)
3. Microinjection embryonic cells into blastocyst
4. Transfer blastocyst to pseudo-pregnant female (hormonal horny mouse)
5. Breed chimeric mouse
6. Breed heterozygous mice
7. Obtain mouse homozygous for gene knockout

Question 6

What is a gene-targeting construct made of?

Answer 6

1. plasmid backbone
2. homology arm x2
3. neo-resistant cassette (inside arms)
4. thymidine kinase cassette (outside arms)
   The gene-targeting construct is inserted into the genome via homologous recombination

Question 7

What is “positive selection” for selection of transfectants?

Answer 7

Cells that didn’t insert the targeting vector will die in a neomycin culture
Those who have will get the neo-resistant gene and live
Also those who have random gene insertion, but then negative selection will kill them

Question 8

What is “negative selection” for selection of transfectants?

Answer 8

Cells that have random gene insertion of the targeting vector will also insert the thymidine kinase => so they will get killed by Gancyclovir culture
Cells that have targeted (correct) gene insertion will not have the thymidine kinase because it is outside of the homologous arms

Question 9

How to visually select transgene mice?

Answer 9

Targeted embryonic stem cells from mouse with white coat color are injected into a blastocyst from mouse with black coat color => obtain mosaic color mouse

Question 10

How to obtain founder (F1) generation knock-out mice after blastocyst transfer?

Answer 10

Chimera mice are back-crossed to confirm germline transmission
This takes 12 generation of back-crossing to obtain pure knockout mice (2-3 years of back-crossing)
Than checked with genotyping (PCR or southern blot)

Question 11

What is conditional knock-outs, what are the benefits and what technique is used?

Answer 11

A method to switch genes on and off in a specific tissue and/or in a specific time
It is possible to overcome embryonic lethality, which is a problem with conventional full knockouts, as well as to study gene effects in only one tissue (organ) of interest
Technique: Cre/LoxP

Question 12

What is Cre/LoxP?

Answer 12

Cre is a recombinase enzyme

LoxP is a inverted repeats flanking a region recognized by Cre

Question 13

What are the 3 Cre/LoxP system-scheme?

Answer 13

1. LoxP sites of same direction on same strand: deletion/integration
2. LoxP sites in same direction on parallel strands: translocation
3. LoxP sites in opposite direction on the same strand: inversion