1. Research Techniques Made Simple: Generation of complete or tissue-specific knockout mice
Lukas Scharfenberger, Tina Hennerici, Gábor Király, Sophie Kitzmüller, Marigje Vernoij, Julia G. Zielinski

2. What is a gene knockout?
Manipulation of the DNA that results in the nonfunctionality or complete loss of the corresponding protein
Whole-body knockout:
knockout of a gene in every cell of the organism
Conditional knockout:
knockout of a gene in specific cell populations or organs

3. Generation of gene-manipulated mice by gene targeting
Isolation of embryonic stem (ES) cells
Isolation of
blastocyst
in vitro cultivation of ES cells
Transfection with targeting construct
neo 2
targeting construct
neo 2
homologous recombination 1 2 3 4
neo
gene knockout
wildtype gene

4. Generation of gene-manipulated mice by gene targeting
Selection with neomycin
Injection of
ES cells into
mouse embryo
Breeding
chimeric
offspring
Implantation into
surrogate mother
knockout
mouse

5. Conditional knockout Cre mouse floxed mouse created by gene targeting
loxP
neo 1 2 3 4
tissue-specific
promoter
crossing

6. Conditional knockout Cre mouse Cre;;genefl/fl floxed mouse Cre
site-specific recombination
loxP
neo 1 2 3 4
degradation
neo 2 3 1 4
Tissue-specific knockout

7. Confirmation of gene knockout
concept of
Avcr1b knockout
genotyping PCR
Tissue-specific
knockout in
K14-Cre;;Avcr1bflox/flox
mouse
Qiu et al., 2011

8. Advantages of a conditional knockout model
Overcome embryonic and early postnatal lethality of whole-body knockout mice
Tissue or organ autonomous analysis of a gene function
The combination of a tissue-specific expressing Cre mouse and floxed gene mouse offers a great variety of mouse models
Commercially available mouse lines save time and space

9. Limitations of a conditional
knockout model
Not every human disease can be investigated by a mouse gene knockout
No temporal control of the gene knockout (inducible KO)
Some diseases have an underlying mechanism that results from overactivity of a gene expression (alternative: knockin model)
Redundancy of related gene products may compensate and thus not reveal the gene function