Manipulating Potassium Channel Expression and Function in Hippocampal Neurons by In Utero Electroporation

Abstract

In utero electroporation (IUE) of plasmid DNA into specific brain regions such as hippocampus and cortex has made it possible to reduce protein expression levels or even replace the endogenous protein with site-directed mutant proteins to reveal important physiological consequences. For example, shRNA can mediate targeted knockdown, and can be complemented by simultaneously expressing the shRNA immune wild-type protein to validate on-target effects, or by expression of an shRNA-immune protein harboring site-specific mutations. More recently, IUE has been adapted to express CRISPR/Cas9 components for targeted gene editing that abolishes protein expression. Utilizing these approaches via IUE results in transfected neurons that are interspersed with non-transfected control neurons in the same brain slices, allowing for direct comparisons. Because IUE is performed late in embryonic development and is confined to a relatively small percentage of neurons, developmental compensatory mechanisms that may compromise gene targeting via germ line transmission are minimized. Thus, IUE presents a powerful opportunity to rapidly and cost-effectively dissect molecular and cellular physiology in the context of the intact brain.

In vivo methods for acute modulation of gene expression in the central nervous system

“…Moreover, both viral vectors and/or IUE were successfully used to introduce optogenetic proteins, chemogenetic receptors or voltage sensors into specific cell-types, cortex layers or brain areas, allowing the study of neural circuits in vivo (Ghitani et al, 2015). Furthermore, CRISPR-Cas9 technology, which allows genome editing, regulation and visualization (Gilbert et al, 2013; Mali et al, 2013; Qi et al, 2013), was recently coupled to IUE to study the role of specific genes in brain development in vivo by knockout (Chen et al, 2015; Cheng et al, 2016; Kalebic et al, 2016; Rannals et al, 2016a, b; Shinmyo et al, 2016; Straub et al, 2014; Wang, 2018) or knock-in (Tsunekawa et al, 2016). CRISPR-Cas9 technology coupled to IUE also enabled the study of the subcellular localization of specific proteins by inserting a sequence for a fluorescent protein into their encoding gene (Mikuni et al, 2016).…”

Recent Genome-Editing Approaches toward Post-Implanted Fetuses in Mice

“…Experiments using in utero gene delivery methods are usually performed on mid- to late-gestational fetuses (from E9–18), probably because they are relatively easier to be identified by visual observation and also can be used in basic research on in utero gene therapy [ 15 ]. All these experiments were based on the opening of the abdominal portion of an anesthetized pregnant animal, exposure of uterine horns, and injection of a solution into a specific site of a fetus [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ] or its surrounding or associated tissues, such as placenta [ 30 , 31 , 32 ], amniotic cavity [ 13 , 14 , 26 , 33 , 34 , 35 ], and the yolk sac (YS) [ 36 ], using a glass micropipette (shown in Figure 1 B). The solution contains viral vectors (including recombinant adeno-associated viruses (rAAVs), lentiviruses, adenoviruses (Ad)), or nonviral vectors (including plasmid DNA).…”