Molecular Cellular Biology Laboratory, Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan.
PLoS One. 2021 Sep 30;16(9):e0258026. doi: 10.1371/journal.pone.0258026. eCollection 2021.
Continuous development of the cerebral cortex from the prenatal to postnatal period depends on neurons and glial cells, both of which are generated from neural progenitor cells (NPCs). Owing to technical limitations regarding the transfer of genes into mouse brain, the mechanisms behind the long-term development of the cerebral cortex have not been well studied. Plasmid transfection into NPCs in embryonic mouse brains by in utero electroporation (IUE) is a widely used technique aimed at expressing transgenes in NPCs and their recent progeny neurons. Because the plasmids in NPCs are attenuated with each cell division, the transgene is not expressed in their descendants, including glial cells. The present study shows that an Epstein-Barr virus-based plasmid (EB-oriP plasmid) is helpful for studying long-term cerebral cortex development. The use of the EB-oriP plasmid for IUE allowed transgene expression even in the descendant progeny cells of adult mouse brains. Combining the EB-oriP plasmid with the shRNA expression cassette allowed examination of the genes of interest in the continuous development of the cerebral cortex. Furthermore, preferential transgene expression was achieved in combination with cell type-specific promoter-driven transgene expression. Meanwhile, introducing the EB-oriP plasmid twice into the same individual embryos during separate embryonic development stages suggested heterogeneity of NPCs. In summary, IUE using the EB-oriP plasmid is a novel option to study the long-term development of the cerebral cortex in mice.
胚胎期到出生后的大脑皮层的持续发育依赖于神经元和神经胶质细胞，它们均由神经前体细胞（NPC）产生。由于将基因转入小鼠大脑的技术限制，大脑皮层的长期发育机制尚未得到很好的研究。通过子宫内电穿孔（IUE）将质粒转染到胚胎小鼠大脑中的 NPC 中，是一种广泛用于在 NPC 及其最近的祖细胞神经元中表达转基因的技术。由于 NPC 中的质粒在每次细胞分裂时都会减弱，因此该转基因不会在其后代（包括神经胶质细胞）中表达。本研究表明，基于 Epstein-Barr 病毒的质粒（EB-oriP 质粒）有助于研究大脑皮层的长期发育。使用 EB-oriP 质粒进行 IUE 可以允许转基因在成年小鼠大脑后代的祖细胞中表达。将 EB-oriP 质粒与 shRNA 表达盒结合使用，可以检查大脑皮层连续发育过程中感兴趣的基因。此外，通过与细胞类型特异性启动子驱动的转基因表达相结合，可以实现转基因的优先表达。同时，在不同的胚胎发育阶段将 EB-oriP 质粒两次引入同一个体胚胎中提示 NPC 的异质性。总之，使用 EB-oriP 质粒的 IUE 是一种研究小鼠大脑皮层长期发育的新选择。
