Inducible, Cell-Targeted Mutations in Mice (Transgenic Core)
PPG investigators use several methods to make genetic mutant mice with increased or decreased levels of a protein of interest within the VTA or NAc specifically. One is the tetracycline gene regulation system, which is a two-gene system (Figure 10). One gene expresses the tetracycline transactivator (tTA), a tetracycline inhibitable transcription factor, under the control of some neural promoter. The other gene expresses the protein of interest under the control of the Tetop promoter, which is activated by tTA. This is an inducible system, because when bitransgenic (double transgenic) mice are maintained on the tetracycline derivative, doxycycline, gene expression is off and can be turned on in adult animals by removal of the doxycycline. This is also a cell-type specific system, because the protein is expressed only where tTA is expressed and this can be controlled by the neural promoter used to drive it. As an example, this system has been used to regulate CREB expression in the NAc (see CREB).
We also use the Cre-loxP system, in which a gene of interest is engineered to contain loxP sites flanking a critical region of the gene. A mouse containing the “floxed” gene is normal, because the loxP sites are silent. Upon expression of the Cre recombinase, an enzyme which removes DNA sequences flanked by loxP sites, that gene is inactivated (Figure 11
). We use three methods for inducing Cre in a region-specific manner in brain. In one, we breed mice containing a floxed gene to mice in which Cre is inducibly expressed under the tetracycline system described above. In the second, we breed them to mice that express modified forms of Cre, which can be activated upon local injection of a chemical in brain. In the third, we use viral vectors encoding Cre to create localized knockouts of the floxed gene. For example, this latter method has been used to knockout BDNF from the VTA specifically (see Brain Derived Neurotrophic Factor). Together, these various approaches enable us to exert powerful control over a stress-regulated protein.