Novel animal models for studying complex brain disorders: BAC-driven miRNA-mediated in vivo silencing of gene expression

Abstract

In schizophrenia, glutamic acid decarboxylase 1 (GAD1) disturbances are robust, consistently observed, cell-type specific and represent a core feature of the disease. In addition, neuropeptide Y (NPY), which is a phenotypic marker of a sub-population of GAD1-containing interneurons, has shown reduced expression in the prefrontal cortex in subjects with schizophrenia, suggesting that dysfunction of the NPY+ cortical interneuronal sub-population might be a core feature of this devastating disorder. However, modeling gene expression disturbances in schizophrenia in a cell type-specific manner has been extremely challenging. To more closely mimic these molecular and cellular human post-mortem findings, we generated a transgenic mouse in which we downregulated GAD1 mRNA expression specifically in NPY+ neurons. This novel, cell type-specific in vivo system for reducing gene expression uses a bacterial artificial chromosome (BAC) containing the NPY promoter-enhancer elements, the reporter molecule (eGFP) and a modified intron containing a synthetic microRNA (miRNA) targeted to GAD1. The animals of isogenic strains are generated rapidly, providing a new tool for better understanding the molecular disturbances in the GABAergic system observed in complex neuropsychiatric disorders such as schizophrenia. In the future, because of the small size of the silencing miRNAs combined with our BAC strategy, this method may be modified to allow generation of mice with simultaneous silencing of multiple genes in the same cells with a single construct, and production of splice-variant-specific knockdown animals.

Incentive Learning Underlying Cocaine-Seeking Requires mGluR5 Receptors Located on Dopamine D1 Receptor-Expressing Neurons

“…A similar approach has been reported previously for interference with other genes in nurse cells (Rao et al, 2006) and, together with a very recent report (Garbett et al, 2010), our findings show that this technique can be used successfully in the brain. Compared with conditional gene deletion this approach has the advantage that it involves only one mouse line and offers the perspective to be used, in modified forms, in other organisms in which targeted mutagenesis is not feasible.…”

Modulation of behavioral networks by selective interneuronal inactivation

“…modified BACs contained an enhanced green fluorescent protein (eGFP) reporter gene and a minigene with a GAD1-targeting miRNA at the start codon of either the mNpy or mCck genes (described previously 16 ). All experiments were conducted in accordance with Vanderbilt Animal Care and Use Committee guidelines.…”

“…To test this, we have developed two transgenic mouse lines that suppress GAD1 expression in CCK+ and NPY+ interneuron populations using novel bacterial artificial chromosome (BAC)-driven miRNA-based silencing technology. 16 To uncover molecular and behavioral consequences of GAD1 deficiency in these two distinct interneuronal cell types, we performed in situ proteomic analyses on brain tissue sections and comprehensive behavioral assessments of these transgenic mice.…”

Neurodevelopment, GABA System Dysfunction, and Schizophrenia

“…In fact, our understanding of the developmental importance of the GABAergic system comes largely from transgenic mouse research. Simply put, while there are no (and perhaps never will be) 'true' rodent models of schizophrenia or 'schizophrenic mice', a combined use of transgenic technology and environmental challenges in rodent models is essential for understanding genes, cognition, and mental disorders Garbett et al, 2010Garbett et al, , 2012Jaaro-Peled et al, 2009;Levitt, 2005;Papaleo et al, 2012;Schmidt et al, 2013;Schmidt and Mirnics, 2012;Smith et al, 2007).…”