Gene delivery in mouse auditory brainstem and hindbrain using in utero electroporation.

Abstract

BackgroundManipulation of gene expression via recombinant viral vectors and creation of transgenic knock-out/in animals has revolutionized our understanding of genes that play critical roles during neuronal development and pathophysiology of neurological disorders. Recently, target-specific genetic manipulations are made possible to perform in combination with specific Cre-lines, albeit costly, labor-intensive and time consuming. Thus, alternative methods of gene manipulations to address important biological questions are highly desirable. In this study, we utilized in utero electroporation technique which involves efficient delivery of hindbrain-specific enhancer/promoter construct, Krox20 into the third ventricle of live mouse embryo to investigate green fluorescent protein (GFP) expression pattern in mouse auditory brainstem and other hindbrain neurons.ResultsWe created a GFP/DNA construct containing a Krox20 B enhancer and β-globin promoter to drive GFP expression in the hindbrain via injection into the third ventricle of E12 to E13.5 mice. Electrical currents were applied directly to the embryonic hindbrain to allow DNA uptake into the cell. Confocal images were then acquired from fixed brain slices to analyze GFP expression in mouse whole brain at different postnatal stages (P6-P21). By using a cell-type specific enhancer as well as region specific injection and electroporation, robust GFP expression in the cerebellum and auditory brainstem but not in the forebrain was observed. GFP expression in calyx of Held terminals was more robust in <P15 compared to >P15 mice. In contrast, GFP expression in MNTB neurons was more prevalent in >P15 compared to <P15. In regards to the relative expression of GFP versus the synaptic marker Vglut1, percentage fluorescence GFP intensity in the calyx was higher in P11 to P15 than P6 to P10 and P16 to P21 groups.ConclusionsTaken together, this technique would potentially allow hindbrain-specific genetic manipulations such as knock-down, knock-in and rescue experiments to unravel critical molecular substrates underpinning functional and morphological remodeling of synapses as well as understanding the pathophysiology of certain neurological disorders targeting not only the auditory brainstem but also other parts of hindbrain, most notably the cerebellum.