The strongest evidence of a protein’s function in its natural environment can be achieved through its genetic disruption in a living organism. By comparing the observed effects to an appropriate control, one can draw conclusions about the natural function of the protein (Iredale 1999). In that way, the overall impact on the organism caused by the lack of the protein can directly be observed and the cell types to which the protein is essential can be identified. Eventual compensatory effects through other proteins and cell types give direct evidence about the proteins’ biological relevance. Because all clones have the same genetic background and protein disruption level, and because usually no additional external treatment is needed to induce the KO, there is no additional influence on the cellular function. In addition, directly observable consequences on vitality, fertility, morbidity or brain function do not have to be translated to a living organism. At the same time, this method is the most time and resource consuming.
2.4.1 Model organisms
Besides many established KO model organisms like the fruit fly Drosophila melanogaster, the nematode C. elegans or the zebrafish Danio rerio, the rodents mouse (Mus musculus) and rat (Rattus norvegicus) are among the preferred KO model organisms due to their close genetic and physiological similarities to humans (Capecchi 1994). They are useful tools to study more complex mammalian physiological systems like for instance the nervous system or the cardiovascular system.
Although neurons are a hallmark of most animals, the complexity of their organization varies strongly (see 2.1). In order to study higher brain functions such as learning, memory, empathy, anxiety and addiction, one is often restricted to use mammals as model systems. In neurobiology,
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30 the mouse model is the preferred one because mice are easier to handle than rats, reproduce fast, and share about 99 % of genes with humans (Capecchi 1994).
2.4.2 Mouse mutagenesis
The earliest model organisms were developed by the selection and breeding of animals with specific traits. In chemical mutagenesis, DNA-damaging chemicals were used to introduce random mutations into the genome. The development of genetic technologies made it possible to produce mouse models with the desired mutations to study specific diseases and gene functions.
In transgenesis a new gene randomly integrates into the genome. The DNA of interest is microinjected into the pronucleus of a zygote, which is then transferred into the uterus/oviduct of a pseudopregnant foster female. The chimeric progenitors can then directly be utilized for line expansion and testing. The relatively fast method is a good way to overexpress a gene of interest. Its disadvantage is due to the limited possibility to control the site of integration of the DNA requiring appropriate controls. The generation of loss-of-function models is possible e. g. by overexpression of dominant-negative proteins or by introducing a gene-trapping cassette.
2.4.3 Homologous recombination
In contrast to transgenesis, homologous recombination has the advantage of allowing the control of the genomic location of DNA integration. The desired DNA sequence is flanked by two large homology arms of at least 2 kb of sequence homology (Melton 2002), increasing the probability of DNA integration by homologous recombination (Lin and Sternberg 1984) and a neomycin selection cassette (Smithies, Gregg et al. 1985). The events of random DNA integration still can occur, but can partly be circumvented by introducing a negative selection cassette such as DTA (diphtheria toxin A) into the targeting vector.
The generation of genetically modified mice by homologous recombination requires the linearized targeting vector DNA to be electroporated into cultivated pluripotent embryonic stem cells (ESCs) (Evans and Kaufman 1981) and the selection for positive ESC clones with their subsequent injection into blastocysts (Bradley, Evans et al. 1984). The latter ones are then implanted into foster females by embryo transfer. Male chimeras are preffered due to the possibility of fast line expansion, leading to the fact that normally male ES cells are used. After manipulation of their genetic information the ESCs are injected into blastocysts and the chimeric offspring is used for line expansion. In contrast to transgenesis, gene targeting by using homologous recombination takes more time and expertise because it requires a more complex design of the targeting vector, the electroporation of cultivated ES cells and the selection of positive clones.
2.4.4 Conditional mutagenesis
Since about 15 % of gene KOs are developmentally lethal (www.genome.gov/12514551), spatiotemporal control of gene KO is desirable. Following the development of the Cre/loxP and the Flp/FRT technologies, it was possible to generate conditional KOs (Dymecki 1996, Sauer 1998). The 34 nt loxP (locus of X-ing over derived from bacteriophage P1) sites can be
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introduced around a critical exon of a gene (Gu, Marth et al. 1994). If the two loxP sites are oriented in the same direction, the recombination results in a circular excision of the intervening sequence by Cre recombinase (Sternberg and Hamilton 1981). Thus, Cre recombinases can be used as molecular switches for the excision or inversion of a genomic sequence of interest (see Figure 14).
Figure 14: Mechanism of the Cre/loxP recombination system. Cre recombinase catalyzes the bidirectional recombination reaction between two palindromic loxP sites. In case of an equal orientation of the loxP sites, recombination results in the circular excision of the intervening sequence. The excision of the intervening sequence is favored over its reinsertion.
As in the Cre/loxP system, the FLP/FRT system makes use of a similar palindromic sequence (FRT) and a FLP recombinase derived from yeast. The combination of both systems allows a wide variety of possibilities in conditional mutagenesis.
2.4.5 Spatiotemporal control of Cre and FLP recombinases
The existence of the Cre/loxP and FLP/FRT systems requires conditional expression or activity of the respective recombinases in order to enable spatiotemporal control of gene KO. Several strategies were used to achieve this goal. Recombinases, which are already expressed in germ cells, lead to a recombination of loxP- or FRT-flanked genomic sequences in the whole organism.
These FLP-deleter (Kranz, Fu et al. 2010) and Cre-deleter (Schwenk, Baron et al. 1995) mice are used for the generation of non-conditional, full KO progeny, for instance. In the tetracycline/doxycycline controlled transcriptional activation system, the expression of Cre can either be induced or turned off upon administration of the antibiotics tetracycline or doxycycline (Utomo, Nikitin et al. 1999).
The tamoxifen inducible system is another way to control Cre recombinase activity (Feil, Wagner et al. 1997, Hayashi and McMahon 2002). In this case, a BAC-transgene or a knock-in-allele carries Cre recombinase, which is fused to the ligand-binding domain of the estrogen receptor (ERT2). By three point mutations, the latter one is rendered unresponsive to its natural ligand estrogen. The Cre-ERT2 fusion protein is constitutively expressed but Cre activity is blocked by the binding to heat shock proteins. Upon administration of the synthetic estrogen derivative
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32 tamoxifen, the heat shock proteins dissociate from the ERT2 domain and allow the activated Cre domain to cut loxP flanked DNA segments.
Another possibility to conditionally knock out genes is by the usage of a specific promoter, which is only active in the cell-type of interest and/or at certain time points to control Cre expression.
The advantage of this system is the fact that no additional treatment with either tamoxifen or doxycycline/tetracycline is necessary, which might impact physiological functions (e. g. estrogen plays a role in the hippocampal memory; tetracycline influences the composition of enteric bacteria) (Woolley 1998, Silva, Mello et al. 2000).
In line with that, many promoters have been used to generate mouse models for brain-specific expression of Cre at different stages of development (Nestin-Cre, Emx-Cre, CamKIIα-Cre).
Nestin-Cre mice were widely used for the Cre-mediated excision of loxP-flanked genes in the central and peripheral nervous systems including neuronal and glial cell precursors (Tronche, Kellendonk et al. 1999). The EMX1-Cre knock-in mouse starts to express the Cre recombinase during the development of the forebrain. Cre is specifically expressed in progenitor cells of pyramidal neurons of the cortex, hippocampus and olfactory bulb (Iwasato, Datwani et al. 2000).
The activity of the murine CamKIIα promoter has also been widely studied and the expression of the CamKIIα gene has been described to be mainly restricted to the postnatal forebrain (Burgin, Waxham et al. 1990, Mayford, Bach et al. 1996). In line with that, several transgenic CamKIIα-Cre mouse models were developed (Tsien, Chen et al. 1996, Minichiello, Korte et al. 1999, Casanova, Fehsenfeld et al. 2001). Nevertheless, for many of these tissue-specific promoter-driven Cre mice, a background expression has been reported in several tissues, with other phenotypes unrelated to Cre activity (Liang, Hippenmeyer et al. 2012, Zhang, Dublin et al. 2013).
As an alternative, Cre can be directly introduced by injection using a viral vector (Anton and Graham 1995).
In addition to in situ hybridization and immunohistochemistry in order to visualize expression of Cre RNA or protein, useful mouse models have been developed as genetic tools to visualize Cre activity. In this case, a reporter gene/cDNA, such as LacZ (Soriano 1999) or GFP (Kawamoto, Niwa et al. 2000) is knocked-in into the ROSA26 locus and flanked by loxP sites but not expressed. Only in presence of Cre, the reporter gene is remodified such that it can be expressed and visualized either by LacZ staining or by fluorescence microscopy.
2.4.6 Genome engineering: new approaches in mutagenesis
The generation of mouse models using ES cell-based approaches is time-consuming and requires specific expertise. Genome editing is a recently discovered alternative type of genetic engineering in which genomic DNA can be modified using engineered nucleases. These methods employ endonucleases capable of inducing double-strand breaks (DSB) at desired locations in the chromosomal DNA. The induced DSBs are repaired through the two major cellular DNA
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damage repair pathways nonhomologous end-joining (NHEJ) or homology-directed repair (HDR), resulting in targeted mutations.
Currently, there are four families of engineered nucleases being used: meganucleases (Epinat, Arnould et al. 2003), zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the CRISPR-Cas system (clustered regularly interspaced short palindromic repeats with the CRISPR associated (Cas) nuclease). ZFNs and TALE nucleases are chimeric proteins composed of sequence-specific DNA-binding modules, which are fused to the non-specific Fok1 nuclease (Carroll 2011, Cermak, Doyle et al. 2011). In the CRISPR-Cas system, an RNA molecule navigates the Cas nuclease to the genomic target site (Wang, Yang et al. 2013).