New biological tools for genetic manipulation of the mouse brain

The site-specific gene expression in the mouse brain is the most crucial issue for genetic studies of brain networks. In our studies, we used different fluorescent proteins (FPs) for monitoring brain anatomy, while for the functional analysis, proteins such as Cre recombinase and the tetracycline-controlled transactivator (tTA) were investigated. From the technical point of view, we attempted both mouse transgenic technology and recombinant adeno-associated virus (rAAV) gene delivery in vivo for transferring functional proteins into specific cell types of the mouse brain. First we analyzed the cell-type specificity of a bacterial artificial chromosome (BAC) transgene. We selected an enhanced green fluorescent protein (EGFP) recombined BAC (from Genesat project) which was supposed to have mitral/tufted cell specific expression in the olfactory bulb. By pronuclear injection of the BAC, different founders were obtained. Out of 41 potential founders, one was mitral cell specific, and two were specific for mitral and tufted cells. Regarding the limitations of cost and time using the mouse transgenic technology, we switched as an alternative approach to the rAAV gene delivery system. For visualizing cells that express the virus-delivered proteins, we applied two strategies: one with tTA/rtTA and its bi-directional responder Ptetbi to express Cre recombinase together with an FP in the specific brain regions. This provided a strong expression level of both Cre and FPs in cultured neurons and in neurons in the brain. For the second strategy, we applied a slightly modified T2A self-cleaving peptide bridge for the quantitative expression of Cre recombinase or tTA/rtTA together with FPs, respectively. By applying the T2A peptide approach, two proteins can be almost equally expressed with one rAAV construct. This opens a new avenue for gene function analysis in the central nervous system. Next we analyzed whether rAAV can be used for the cell-type-specific expression. We selected glia cells, since in the transgenic field, specific promoters are described for proteolipidprotein (PLP) and glial fibrillary acidic protein (GFAP). As second cell population we analyzed promoters for local interneurons, the glutamate decarboxylase 67 (GAD67) and the cholecystokinin (CCK). We investigated a complementation approach which splits Cre recombinase into two parts (N-Cre and C-Cre), each driven by a different promoter. The Cre recombinase is active when N-Cre and C-Cre are expressed in the same cell. With this genetic complementation approach, the infected Cre positive cells could be defined as PLP and GFAP or GAD67 and CCK double positive cells, respectively. Thus, the cell-type-specific expression is achieved via rAAV delivery, and the double positive cells for two different promoters are illustrated with the Cre complementation approach. BAC transgenic technology, rAAV gene in vivo delivery, tTA/rtTA inducible gene activation, 2A peptide cleavage approach and the Cre complementation system, all these newly developed biological tools can be taken advantage of different aspects for different experimental purposes. Moreover, they open a convenient avenue for site-specific and cell-type-specific gene expression in manipulating brain circuits.