One major limitation of in utero gene transfer technique was that modulation of gene expression may not be precisely temporally controlled. As we discussed above, many genetic factors for schizophrenia have roles at different timings during neurodevelopment (Harrison and Weinberger, 2005; Jaaro-Peled et al., 2009), such that DISC1 has roles in cell proliferation, neuronal migration, and dendritic development/spine formation in the developing cerebral cortex as well as synapse function in cerebral cortex and positioning of newborn neurons in the dentate gyrus at the adult stage (Duan et al., 2007; Jaaro-Peled et al., 2009; Kamiya et al., 2005; Mao et al., 2009). Therefore, it is critical to segregate each molecule's functions in the developmental stages as well as the adult stage in order to address these mechanisms precisely. To overcome this limitation, an inducible gene expression system has been recently applied to in utero gene transfer (Matsuda and Cepko, 2007). In this system, a plasmid construct in which Cre recombinase is expressed in response to intraperitoneally injected 4-hydroxytamoxifen (4-OHT) and a target gene or microRNAs expression construct where a stop codon flanked by two loxP sites downstream from the promoter region is contained, are co-electroporated into embryonic brains. Expression of target gene or microRNAs is induced by tamoxifen-mediated Cre recombination mechanism. By utilizing this system, LoTurco and colleagues reported that conditional re-expression of doublecortin (DCX) at P0 normalized impaired neuronal migration elicited by the conventional knockdown of DCX at E14 (Manent et al., 2009). It is noteworthy that re-expression of DCX ameliorated the susceptibility to convulsant-induced seizures in DCX knockdown animal at P30, supporting the utility of in utero gene transfer not only for studying on the functional role of target gene in molecular and anatomical levels in brain development, but also for examining how genetic deficits in developmental stages may affect brain functions.
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