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

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

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

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

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.

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 www.genoway.com

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

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

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

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

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. 1995 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. 2009 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)

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

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

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

Thanks for your attention !