Multiple events of gene manipulation via in pouch electroporation in a marsupial model of mammalian forebrain development

• Published in: Journal of neuroscience methods 2018-01, Vol.293, p.45-52
• Main Authors: Paolino, Annalisa, Fenlon, Laura R., Kozulin, Peter, Richards, Linda J., Suárez, Rodrigo
• Format: Article
• Language: English
• Subjects: Anesthesiology - instrumentation; Animal model; Animals; Brain development; Brain evolution; Electrodes; Electroporation; Electroporation - methods; Equipment Design; Gene Expression Regulation, Developmental; Genetic Vectors - administration & dosage; Immunohistochemistry; Marsupialia - growth & development; Microscopy, Fluorescence; Models, Animal; Neuronal labelling; Neurons - cytology; Prosencephalon - cytology; Prosencephalon - growth & development; Survival Analysis; Transgenesis
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2017.09.004

[Display omitted] •In pouch electroporation allows several transfection events in the same animal.•Minimally-invasive procedure with high success and spatiotemporal accuracy.•Enhanced access to manipulate distinct brain circuits independently.•Marsupial neuron transgenesis in vivo enables molecular studies of forebrain evolution. The technique of in utero electroporation has been widely used in eutherians, such as mice and rats, to investigate brain development by selectively manipulating gene expression in specific neuronal populations. A major challenge, however, is that surgery is required to access the embryos, affecting animal survival and limiting the number of times it can be performed within the same litter. Marsupials are born at an early stage of brain development as compared to eutherians. Forebrain neurogenesis occurs mostly postnatally, allowing electroporation to be performed while joeys develop attached to the teat. Here we describe the method of in pouch electroporation using the Australian marsupial fat-tailed dunnart (Sminthopsis crassicaudata, Dasyuridae). In pouch electroporation is minimally invasive, quick, successful and anatomically precise. Moreover, as no surgery is required, it can be performed several times in the same individual, and littermates can undergo independent treatments. As compared to in utero electroporation in rodents, in pouch electroporation in marsupials offers unprecedented opportunities to study brain development in a minimally invasive manner. Continuous access to developing joeys during a protracted period of cortical development allows multiple and independent genetic manipulations to study the interaction of different systems during brain development. In pouch electroporation in marsupials offers an excellent in vivo assay to study forebrain development and evolution. By combining developmental, functional and comparative approaches, this system offers new avenues to investigate questions of biological and medical relevance, such as the precise mechanisms of brain wiring and the organismic and environmental influences on neural circuit formation.