Journal of Visualized Experiments www.jove.com Copyright © 2013 Journal of Visualized Experiments September 2013 | 79 | e50146 | Page 1 of 13 Video Article Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening Sandra Vomund 1 , Tamar Sapir 2 , Orly Reiner 2 , Maria A. de Souza Silva 3 , Carsten Korth 1 1 Department of Neuropathology, Medical School Düsseldorf 2 Department of Molecular Genetics, Weizmann Institute for Science 3 Center of Behavioral Neuroscience, University of Düsseldorf Correspondence to: Carsten Korth at ckorth@uni-duesseldorf.de URL: https://www.jove.com/video/50146 DOI: doi:10.3791/50146 Keywords: Neuroscience, Issue 79, Hippocampus, Memory, Schizophrenia, In utero electroporation, in vivo bioluminescence imaging, Luciferase, Disrupted-in-schizophrenia-1 (DISC1) Date Published: 9/24/2013 Citation: Vomund, S., Sapir, T., Reiner, O., de Souza Silva, M.A., Korth, C. Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening. J. Vis. Exp. (79), e50146, doi:10.3791/50146 (2013). Abstract In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at ≥6 months after birth. We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience. Video Link The video component of this article can be found at https://www.jove.com/video/50146/ Introduction The development of the in utero electroporation (IUE) method which allows a modulation of gene expression in the developing brain, has been a break-through since it enabled studying neurodevelopment with relative ease. 1-7 Changes in expression levels of a target gene in a specific brain region during embryonic and/or perinatal development in rodents were demonstrated to critically influence neuronal proliferation, migration, arborization, and connectivity. 8-10 Schizophrenia is a complex mental illness with acute and chronic symptoms that has been related to neurodevelopmental abnormalities 11, 12 and therefore many of the identified candidate genes for schizophrenia are investigated for potential modulating effects on neurodevelopment, like for example for the disrupted-in-schizophrenia-1 (DISC1) gene 13-15 . Brain development is regulated by genetic factors and their interactions with environment which play roles in pre-, peri- and postnatal developmental periods. One major genetic risk factor for various behavioral disorders is the DISC1 16 gene. DISC1 knockdown leads to migration defects in mice 13, 17 , and manipulation of DISC1 expression in the developing cortex by IUE has been shown to impact behavior of adult mice 18 . Manipulating brain gene expression by IUE has several advantages 19 over the generation of transgenic animal lines. First, gene expression within areas of interest is achieved within weeks to months rather than several generations of breeding transgenic rodent lines. Second, compensatory mechanisms during early development that may shield phenotypes in germline-engineered animals 20 are avoided. Third, through