1. Aina Martin Mehods Seminar 03.12.14
Knock out Mouse
Aina Martin
Mehods Seminar

2. History
Mario R. Capecchi, Martin J. Evans and Oliver Smithies

3. Why to use a knock out mouse?
It is a genetically engineered mouse in which one or more genes have been turned off
It´s important for study the role of genes
Reveals the mechanisms of disease and novel therapies
Pre-KO knowlege
SP-C +/ SP-C -/-
Mouse KO knowlege
SP-C -/ rSPC.SP-C -/-
Therapy in KO
Potential therapy
Studing the role of genes: we know the sequence but not the function
Function: Jo Rae, Nat Rev 2005
Histo pics: Botas C Proc Natl Sci 1998
Therapy in KO: Whitsett Am. J. Physiol. Lung. Cell 2000
Potential therapy for astma, COPD, neonathal chronic LD, cystic fibrosis: Clark, immunobiol. 2002
The potential of recombinant Sp-C threapy to reduce lung inflammation

4. Forward and reverse genetic modification in mice
KNOCK OUT
Gene alteration
Study the phenotype
ADVANTAGE
Find out which genes play a role in the disease
LIMITATION
The assumption may be wrong and we have to start again

5. Basic strategies to generate a knock out
The true knock out needs both copies of the gene!
Basic strategy in generating muse models of knockout genes
Take totipotent ES that we will modificate and select
Introduce again into the blastocyst where they will differentiate into all cell types as normally to create a chimera  the ES are acting as vehicle to introduce the manipulated genetic material
The positive selection is achieved by introducing a neomycin resistance gene (neor) into the centre of the target gene  only those ES containing the resistane will grow in the cond medium
We also have to get read off those cells that have incorporated the plasmid not at the expected complementary but at some other part of the genome by random integration  with the hepes virus derived thymidine kinase sequence, only the ones expressing the protein will dye in presence of ganciclovir
At the end those cells that survives in the neomycin medium will survived but they will dye if they have been introduced in the wrong place.

6. Constitutive knock out
Target gene is permanently disrupted or inactivated in every cell
 Covers all stages of development (from the 1-cell embryo through adulthood)
Fast and cost-effective solution for the preliminary study of target gene function in vivo
May be embryonic lethal and is likely to result in the alteration of other genes expression
Conventional gene
knockout ablates a gene in all cells, usually resulting in complex
phenotypes, or embryonic lethality if the gene product is critical in
development.
This system consists of
a 38 kD Cre DNA recombinase and two 34 bp loxP sites flanking
a target DNA sequence. The Cre recombinase recognizes loxP
sites and excises the target DNA sequence when the orientation of
two loxP sites is cis-repeated [1]. For a tissue-specific gene
knockout strategy, the Cre coding sequence is usually driven by
a tissue-specific promoter, which allows Cre to be expressed only
in one type of tissue. Thus, deletion of the gene of interest also
occurs only in the tissue where Cre is expressed
Cre system picture = mattias Zepper, own work

7. Conditional knock out
Restrict the targeting of the floxed gene to a particular organ or a particular time
Avoids complex pehonypes or embryonic lethality if the gene is critical in development
Requires previous analysis to avoid endogenous gene deregulation or truncated protein expression.
The timeline the design is longer
Conventional gene
knockout ablates a gene in all cells, usually resulting in complex
phenotypes, or embryonic lethality if the gene product is critical in
development.
This system consists of
a 38 kD Cre DNA recombinase and two 34 bp loxP sites flanking
a target DNA sequence. The Cre recombinase recognizes loxP
sites and excises the target DNA sequence when the orientation of
two loxP sites is cis-repeated [1]. For a tissue-specific gene
knockout strategy, the Cre coding sequence is usually driven by
a tissue-specific promoter, which allows Cre to be expressed only
in one type of tissue. Thus, deletion of the gene of interest also
occurs only in the tissue where Cre is expressed
Cre system picture = mattias Zepper, own work

8. Conditional knock out Tissue-specific Knockout Mouse
Cre-loxP recombination system using a tissue-specific promotor
Example:
Type II cell-specific VEGF knockout mouse
Marco Maura et al. Am. J. Pathol. 2010
Conventional gene
knockout ablates a gene in all cells, usually resulting in complex
phenotypes, or embryonic lethality if the gene product is critical in
development.
This system consists of
a 38 kD Cre DNA recombinase and two 34 bp loxP sites flanking
a target DNA sequence. The Cre recombinase recognizes loxP
sites and excises the target DNA sequence when the orientation of
two loxP sites is cis-repeated [1]. For a tissue-specific gene
knockout strategy, the Cre coding sequence is usually driven by
a tissue-specific promoter, which allows Cre to be expressed only
in one type of tissue. Thus, deletion of the gene of interest also
occurs only in the tissue where Cre is expressed
Cre system picture = mattias Zepper, own work

9. Conditional knock out Inducible Knockout Mouse
To study at various defined developmental ages or stages
To model age-related diseases
Cre-loxP recombination system with temporal-specific activity
rtTA/TetO-Cre (Doxocyclin system)
Promotor
Gen of int F0 F1 x F3
Activator line
Operator line
Example:
P1 bacteriophage recombinase (Cre) to direct site-specific DNA recombination between pairs of LoxP sites
The tetracycline (tet) inducible system was developed independently to the Cre-LoxP system
and has different advantages and limitations. The tet system
in vivo
consists of two
transgenic mouse lines, an activator line and an operator line. The activator line expresses
either the tetracycline activator (TA, tet-off) or the reverse tetracycline responsive
transactivator (rtTA, tet-on) in a tissue specific manner. The operator line carries a transgene
of interest under control of the (tetO)7CMV operator (tetO). In double transgenic mice,
doxycycline either causes the tTA to bind the tetO sequence, suppressing transcription (Tet-
off), or causes the rtTA to bind to the tetO sequence, activating transcription of the gene of
interest (Tet-on)
CreERT2 fusion protein is cytoplasmic. Upon binding to tmx, CreERT2 translocates to the nucleus where it accesses the LoxPsites
Cre recombination was acti-
vated by dox for 2 or 10 days to induce activation of DT-A causing
acute or chronic Clara cell ablation
Clara cell secretory protein (CCSP
Cre-recombinase activity is controlled by doxycycline temporally

10. Conditional knock out Inducible Knockout Mouse
To study at various defined developmental ages or stages
To model age-related diseases
Cre-loxP recombination system with temporal-specific activity
Using a combination with the LacZ reporter allow us to see which cells have been transfected (X-gal stainig)
Example:
Cre-ERT2 (Tamoxifen system)
Citoplasmatic
Cre-ERT2
+tmx
Nucleus
P1 bacteriophage recombinase (Cre) to direct site-specific DNA recombination between pairs of LoxP sites
The tetracycline (tet) inducible system was developed independently to the Cre-LoxP system
and has different advantages and limitations. The tet system
in vivo
consists of two
transgenic mouse lines, an activator line and an operator line. The activator line expresses
either the tetracycline activator (TA, tet-off) or the reverse tetracycline responsive
transactivator (rtTA, tet-on) in a tissue specific manner. The operator line carries a transgene
of interest under control of the (tetO)7CMV operator (tetO). In double transgenic mice,
doxycycline either causes the tTA to bind the tetO sequence, suppressing transcription (Tet-
off), or causes the rtTA to bind to the tetO sequence, activating transcription of the gene of
interest (Tet-on)
CreERT2 fusion protein is cytoplasmic. Upon binding to tmx, CreERT2 translocates to the nucleus where it accesses the LoxPsites
Mark Lewandoski, Nat. Rev. Genetics 2001

11. Limitations Inducible Knockout Mouse LIMITATIONS !!
DOXOCYCLIN SYSTEM
Doxycycline is a matrix metalloproteinase inhibitor and has been shown to promote pulmonary hypertension after hypoxia, and to attenuate mucin production (Perl AK, Proc Natl Acad Sci 2002 and Am J Respir Cell Mol Biol. 2005)
Thracheal Clara cells are sensitive to doxycycline treatment (Smith RW, Hicks DA, Am. J. Respir. Cell Mol. Biol. 2011)
Single transgenic mice, and mice containing all transgenes, should be tested in the absence of doxycycline
Doxycycline is stored in tissues and will be released over time  timing and duration of dose need to be determined!
*to target subsets of lung epithelial cells, it´s described (Sadowski PD. Res Mol Biol and Farley FW, et al. Genesis. 2000)
TAMOXIFEN SYSTEM
Administration of tmx or 4OH-T by itself can be toxic 8Hayashi S; Dev Biol. 2002)
and can cause a transient increase in blood pressure in adult (Wirth A, Nat Med. 2008)
CRE-LoxP SYSTEM
Toxic effects of the Cre recombinase (probably due to endogenous cryptic LoxP) have been reported in the lung (Perl AK, Am J Respir Cell Mol Biol and Smith RW, 2011)
P1 bacteriophage recombinase (Cre) to direct site-specific DNA recombination between pairs of LoxP sites
The tetracycline (tet) inducible system was developed independently to the Cre-LoxP system
and has different advantages and limitations. The tet system
in vivo
consists of two
transgenic mouse lines, an activator line and an operator line. The activator line expresses
either the tetracycline activator (TA, tet-off) or the reverse tetracycline responsive
transactivator (rtTA, tet-on) in a tissue specific manner. The operator line carries a transgene
of interest under control of the (tetO)7CMV operator (tetO). In double transgenic mice,
doxycycline either causes the tTA to bind the tetO sequence, suppressing transcription (Tet-
off), or causes the rtTA to bind to the tetO sequence, activating transcription of the gene of
interest (Tet-on)
CreERT2 fusion protein is cytoplasmic. Upon binding to tmx, CreERT2 translocates to the nucleus where it accesses the LoxPsites
! The rtTA toxicity in SPC-rtTA mice has been reported to impaired alveologenesis, abnormal expression of surfactant-associated proteins, and mouse death (Morimoto M, Dev Biol 2009 and Whitsett JA, Am J Respir Cell Mol Biol 2006)

12. Conditional knock out Split-Cre system Flipase system (FLP-FRT)
Allows CRE expression only when the activity of two promoters are present
Increases specificity of Cre-mediated DNA recombination
*combination with Cre-ERT2 is also possible
Overcomes Cre-system limitations using a flipase recombinase
BUT no specific lung flipase has been generated yet
Flipase system (FLP-FRT)
Flipase
Gene of int
FRT x
Flip
P1 bacteriophage recombinase (Cre) to direct site-specific DNA recombination between pairs of LoxP sites
The tetracycline (tet) inducible system was developed independently to the Cre-LoxP system
and has different advantages and limitations. The tet system
in vivo
consists of two
transgenic mouse lines, an activator line and an operator line. The activator line expresses
either the tetracycline activator (TA, tet-off) or the reverse tetracycline responsive
transactivator (rtTA, tet-on) in a tissue specific manner. The operator line carries a transgene
of interest under control of the (tetO)7CMV operator (tetO). In double transgenic mice,
doxycycline either causes the tTA to bind the tetO sequence, suppressing transcription (Tet-
off), or causes the rtTA to bind to the tetO sequence, activating transcription of the gene of
interest (Tet-on)
CreERT2 fusion protein is cytoplasmic. Upon binding to tmx, CreERT2 translocates to the nucleus where it accesses the LoxPsites

13. How to prove that your mouse is a true knock out
Conventional gene
knockout ablates a gene in all cells, usually resulting in complex
phenotypes, or embryonic lethality if the gene product is critical in
development.
This system consists of
a 38 kD Cre DNA recombinase and two 34 bp loxP sites flanking
a target DNA sequence. The Cre recombinase recognizes loxP
sites and excises the target DNA sequence when the orientation of
two loxP sites is cis-repeated [1]. For a tissue-specific gene
knockout strategy, the Cre coding sequence is usually driven by
a tissue-specific promoter, which allows Cre to be expressed only
in one type of tissue. Thus, deletion of the gene of interest also
occurs only in the tissue where Cre is expressed
Cre system picture = mattias Zepper, own work

14. Guess..!
Which is the gene of interest??
Is it a constitutive or inducible system??
Which cell line does the promotor belong?
Why did they prove the knockout with a
X-gal staining?
P1 bacteriophage recombinase (Cre) to direct site-specific DNA recombination between pairs of LoxP sites
The tetracycline (tet) inducible system was developed independently to the Cre-LoxP system
and has different advantages and limitations. The tet system
in vivo
consists of two
transgenic mouse lines, an activator line and an operator line. The activator line expresses
either the tetracycline activator (TA, tet-off) or the reverse tetracycline responsive
transactivator (rtTA, tet-on) in a tissue specific manner. The operator line carries a transgene
of interest under control of the (tetO)7CMV operator (tetO). In double transgenic mice,
doxycycline either causes the tTA to bind the tetO sequence, suppressing transcription (Tet-
off), or causes the rtTA to bind to the tetO sequence, activating transcription of the gene of
interest (Tet-on)
CreERT2 fusion protein is cytoplasmic. Upon binding to tmx, CreERT2 translocates to the nucleus where it accesses the LoxPsites

15. Thanks for your attention !