Abstract
BackgroundGene knockdown analyses using the in utero electroporation method have helped reveal functional aspects of genes of interest in cortical development. However, the application of this method to analyses in later stages of brain development or in the adult brain is still difficult because the amount of injected plasmids in a cell decreases along with development due to dilution by cell proliferation and the degradation of the plasmids. Furthermore, it is difficult to exclude the influence of earlier knockdown effects.Methodology/Principal FindingsWe developed a tightly controlled conditional knockdown system using a newly constructed vector, pT2K-TBI-shRNAmir, based on a Tol2 transposon-mediated gene transfer methodology with the tetracycline-inducible gene expression technique, which allows us to maintain a transgene for a long period of time and induce the knockdown of the gene of interest. We showed that expression of the endogenous amyloid precursor protein (APP) was sharply decreased by our inducible, stably integrated knockdown system in PC12 cells. Moreover, we induced an acute insufficiency of Dab1 with our system and observed that radial migration was impaired in the developing cerebral cortex. Such inhibitory effects on radial migration were not observed without induction, indicating that our system tightly controlled the knockdown, without any expression leakage in vivo.Conclusions/SignificanceOur system enables us to investigate the brain at any of the later stages of development or in the adult by utilizing a knockdown technique with the aid of the in utero electroporation gene transfer methodology. Furthermore, we can perform knockdown analyses free from the influence of undesired earlier knockdown effects.
Highlights
During the development of the cerebral cortex, neurons are generated in the ventricular zone (VZ) and migrate outward to the cortical plate (CP), an event that is called ‘‘radial migration.’’ These neurons stop their movement and settle into the six layers of the cortex in an inside-out pattern, whereby early-born neurons are positioned in the deeper layers, and later-born neurons are located in the more superficial layers, migrating outward by passing the earlier born neurons [1]
Layer-specific stereotyped projection patterns are established for each layer of the cortex: layer V neurons, which are born around embryonic day (E)12.5, mainly project to the subcortical regions as the subcortical projection, whereas layer II/III pyramidal neurons, which are born around E14.5, mainly project to the contralateral cortex as the callosal projection [2,3]. In such studies of cortical development, in utero electroporation gene transfer is frequently employed because it enables us to perturb the in vivo gene expression in a convenient manner compared to conventional techniques, such as making genetically modified animals [4,5]
Using the in utero electroporation gene transfer, it has been revealed that various molecules are involved in the development of the cerebral cortex by controlling radial migration
Summary
During the development of the cerebral cortex, neurons are generated in the ventricular zone (VZ) and migrate outward to the cortical plate (CP), an event that is called ‘‘radial migration.’’ These neurons stop their movement and settle into the six layers of the cortex in an inside-out pattern, whereby early-born neurons are positioned in the deeper layers, and later-born neurons are located in the more superficial layers, migrating outward by passing the earlier born neurons [1].In the later stage of the cortical development, just after radial migration ends, neurons that have settled in the cortex continue to elongate their axons toward their targets. For the purpose of conditional gene knockdown at any point during development, we constructed the inducible knockdown vector pT2K-TBI-shRNAmir based on the Tol2 transposable vector pT2K-BI-TRE-EGFP by inserting the mir30 cassette (shRNAmir) and exchanging the promoter (Figure 1A). PT2K-CAGGS-rtTA-M2, which expresses the modified reverse tetracycline-controlled transactivator (rtTA-M2) [22], and pT2KTBI-shRNAmir are designed such that Tol2-flanking regions are integrated into the chromosome in the presence of Tol2 transposase.
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